Stabilized pharmaceutical formulations of insulin analogues and/or insulin derivatives

ABSTRACT

Stabilized pharmaceutical formulations of insulin analogues and/or insulin derivatives are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/002,063, filed Aug. 25, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/726,586, filed Oct. 6, 2017, which is acontinuation of U.S. patent application Ser. No. 14/172,151, filed Feb.4, 2014, now U.S. Pat. No. 9,839,675, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/761,434, filed Feb. 6, 2013,and European Patent Application No. 13305126.8, filed Feb. 4, 2013, theentire contents of which are incorporated by reference herein.

INTRODUCTION

The present invention relates to a pharmaceutical formulation of atleast one insulin analogue and/or insulin derivative, a process forpreparing the pharmaceutical formulation of at least on insulin analogueand/or insulin derivative, and a related kit. It also relates to thepharmaceutical formulation of at least one insulin analogue and/orinsulin derivative and to the related kit for use in the treatment ofdiabetes mellitus, hyperglycemia, and/or for use in lowering bloodglucose levels. The present invention also relates to the use of amedical device for administering the pharmaceutical formulation of atleast one insulin analogue and/or insulin derivative to an animal and/orhuman.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a metabolic disorder in which the ability toutilize glucose is more or less completely lost.

For decades, insulin has been used in the treatment of diabetesmellitus. Several insulin formulations have been developed, e.g. insulinzinc (Zn (II)) suspension, formulations containing protamine, etc.Further, the active pharmaceutical ingredient insulin itself has beenmodified by developing fast acting insulin analogues (e.g. insulinaspart, insulin lispro, insulin glulisine) and long acting insulinanalogues and derivatives (e.g. insulin detemir, insulin degludec,insulin glargine). Fast acting insulin preparations are usuallysolutions of insulin, while long acting insulin preparations can besuspensions containing insulin in crystalline and/or amorphous formprecipitated by the addition of zinc (Zn(II)) salts (e.g. zinc chloride)alone or by addition of protamine or by a combination of both.

The chemical and physical stability of insulin formulations is veryimportant. Insulin formulations are often administered by using peninjection devices or insulin pumps in which an insulin formulation isstored in cartridges until the entire cartridge is empty. Insulinformulations may also be stored in vials, requiring a stable formulationwith respect to chemical and physical stability across the shelf life ofthe formulation.

The chemical and/or physical stability of insulin, insulin analoguesand/or insulin derivatives strongly depends on the pharmaceuticalformulation, e.g. the solvent, the pH value and the excipients. Brangeet al. (Acta Pharm. Nord. 4(3), pp. 149-158, 1992) disclose severalaspects in connection with the chemical stability of insulin. WO2004/080480 discloses pharmaceutical preparations comprisingacid-stabilized insulin. GB 835,638 discloses insulin crystalsuspensions having a protracted effect. WO 98/56406 discloses stableinsulin formulations. U.S. Pat. No. 6,489,292 discloses stable aqueousinsulin preparations without phenol and cresol. U.S. Pat. No. 6,211,144discloses stable concentrated insulin preparations for pulmonarydelivery. Bhatt et al. (Pharmaceutical Research, Vol. 7, No. 6, pp.593-599, 1990) disclose chemical pathways of peptide degradation. Patelet al. (Pharmaceutical Research, Vol. 7, No. 7, pp. 703-711, 1990)disclose chemical pathways of peptide degradation. Tyler-Cross et al.(Journal of Biological Chemistry, Vol. 266, No. 33, Issue of November25, pp. 22549-22556, 1991) disclose effects of amino acid sequence,buffers, and ionic strength on the rate and mechanism of deamidation ofasparagine residues in small peptides. GB 840,870 discloses improvementsin or relating to insulin preparations. U.S. Pat. No. 6,852,694discloses stabilized insulin formulations. Galloway et al. (Diabetes—TheJournal of the American Diabetes Association, Vol. 21, No. Suppl. 2, pp.637-648, 1972) disclose new forms of insulin. Jackson et al. discloseseveral aspects with regard to neutral regular insulin (Diabetes—TheJournal of the American Diabetes Association, Vol. 21, No. 4, pp.235-245, 1972). Lill (Pharmazie in unserer Zeit, No. 1, pp. 56-61, 2001)discloses general aspects in connection with insulin formulations. TheGerman product specification of the medicinal product Berlinsulin® HNormal 3 mL Pen discloses a formulation containing human insulin,metacresol, glycerol, water and optionally hydrochloric acid and sodiumhydroxide. The German product specification of the medicinal productActrapid® discloses a formulation containing human insulin, zincchloride, glycerol, metacresol, sodium hydroxide, hydrochloric acid andwater.

The solubility of insulin, insulin analogues and/or insulin derivativesin aqueous media depends on the pH value. For example, the lowestsolubility is shown close to the isoelectric point which for humaninsulin is around pH 5.3 and 5.4. Very good solubility can be observedat pH values below 4 and above 7. However, insulin suffers fromdegradation at strong acidic conditions and strong alkaline conditions.Therefore, most of the medicinal products containing insulin, insulinanalogues and/or insulin derivatives have a pH value in the range of 7.2to 7.4 and mostly buffering agents are used to achieve and maintain thepH within this range.

It has now surprisingly been found that an alternative aqueouspharmaceutical formulation comprising at least one insulin analogueand/or insulin derivative comprising sodium chloride, without anyadditional buffering agent, shows an excellent chemical and physicalstability, which qualifies this aqueous pharmaceutical formulation as amedicinal product having a defined shelf life.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a pharmaceuticalformulation comprising

(a). at least one analogue and/or derivative of insulin; and

(b). Zn(II); and

(c). sodium chloride; and

(d). optionally protamine;

wherein the pharmaceutical formulation has a pH value in the range offrom 6.0 to 9.0 and is free of any additional buffering agent.

In another embodiment, the pharmaceutical formulation according to thepresent invention is an aqueous pharmaceutical composition.

In another embodiment, the pharmaceutical formulation according to thepresent invention does not contain any additional buffering agent.

In another embodiment, the pharmaceutical formulation according to thepresent invention does not contain any additional buffering agent otherthan the at least one analogue and/or derivative of insulin and theoptionally present protamine.

In another embodiment, the pharmaceutical formulation according to thepresent invention is free of any additional buffering agent selectedfrom the group consisting of 2-amino-2-hydroxymethyl-propane-1,3-diol(TRIS), phosphate, citric acid or citrate salts, acetic acid and saltsthereof, glycylglycine and methionin.

In another embodiment, the pharmaceutical formulation according to thepresent invention does not contain any additional buffering agentselected from the group consisting of 2-aminohydroxymethyl-propane-1,3-diol (TRIS), phosphate, citric acid or citratesalts, acetic acid and salts thereof, glycylglycine and methionin.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises at least one analogue and/or derivative ofinsulin and optionally protamine, wherein the only componentcontributing any buffering activity is the at least one analogue and/orderivative of insulin and optionally protamine.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises at least one analogue and/or derivative ofinsulin and optionally protamine, wherein the total concentration of anybuffering agent in the present invention is in the range from 3.496mg/mL (rounded: 3.5 mg/mL) to 3.996 mg/mL (rounded: 4.0 mg/mL), from3.496 mg/mL (rounded: 3.5 mg/mL) to 3.816 mg/mL (rounded: 3.8 mg/mL) orthe total concentration of any buffering agent in the present inventionis 3.496 mg/mL (rounded: 3.5 mg/mL).

In another embodiment, the pharmaceutical formulation according to thepresent invention consists of (a). at least one analogue and/orderivative of insulin; and (b). Zn(II); and (c). sodium chloride; and(d). optionally protamine; and (e). metacresol; (f). phenol; and (g).polysorbate 20;

and (h). sodium hydroxide and/or hydrochloric acid for pH adjustment toa pH value in the range of from 6.0 to 9.0; and (i). water.

In another embodiment, the pharmaceutical formulation according to thepresent invention is an aqueous pharmaceutical formulation.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises at least one analogue and/or derivative ofinsulin which has or have an isoelectric point (IEP) in the range from4.0 to 6.0, from 4.5 to 6.0, from 4.5 to 5.5, from 5.0 to 5.5, from 5.0to 5.2 or 5.1.

In another embodiment, the pharmaceutical formulation according to thepresent invention has a pH value in the range from 6.5 to 8.5, in therange from 7.0 to 8.0, from 7.0 to 7.8, from 7.1 to 7.6, 7.2, 7.3, 7.4or 7.5, or 7.4.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises at least one analogue of insulin which isselected from the group consisting of insulin aspart, insulin lisproand/or insulin glulisine. In one embodiment, the pharmaceuticalformulation according to the present invention comprises an analogue ofinsulin which is insulin lispro. In one embodiment, the pharmaceuticalformulation according to the present invention comprises an analogue ofinsulin which is insulin aspart. In one embodiment, the pharmaceuticalformulation according to the present invention comprises an analogue ofinsulin which is insulin glulisine.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises a derivative of insulin which is selectedfrom the group consisting of insulin detemir and/or insulin degludec. Inone embodiment, the pharmaceutical formulation according to the presentinvention comprises a derivative of insulin which is insulin detemir. Inone embodiment, the pharmaceutical formulation according to the presentinvention comprises a derivative of insulin which is insulin degludec.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises at least one analogue and/or derivative ofinsulin which is present in a concentration from 10 U/mL to 1000 U/mL,from 10 U/mL to 600 U/mL, from 10 U/mL to 300 U/mL, from 50 U/mL to 300U/mL or 100 U/mL.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises an analogue and/or derivative of insulinwhich is present in a concentration from 60 to 6000 nmol/mL, from 60nmol/mL to 3600 nmol/mL, from 60 nmol/mL to 1800 nmol/mL, from 300nmol/mL to 1800 nmol/mL or 600 nmol/mL.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises Zn(II) which is present in a concentrationfrom 0.0100 mg/mL to 0.0600 mg/mL, from 0.0150 mg/mL to 0.0500 mg/mL,from 0.0150 mg/mL to 0.0300 mg/mL, from 0.0150 mg/mL to 0.0200 mg/mL,from 0.0190 mg/mL to 0.0200 mg/mL, or 0.0196 mg/mL.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises Zn(II) which is present in a concentrationfrom 0.0100 mg/100 U to 0.0600 mg/100 U, from 0.0150 mg/100 U to 0.0500mg/100 U, from 0.0150 mg/100 U to 0.0300 mg/100 U, from 0.0150 mg/100 Uto 0.0200 mg/100 U, from 0.0190 mg/100 U to 0.0200 mg/100 U, or 0.0196mg/100 U.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises sodium chloride which is present in aconcentration from 0.01 mg/mL to 15 mg/mL, from 0.1 mg/mL to 15 mg/mL,from 0.1 mg/mL to 10 mg/mL, from 1 mg/mL to 10 mg/mL, from 2.0 mg/mL to10 mg/mL, from 3.0 mg/mL to 9.0 mg/mL, from 4.0 mg/mL to 9.0 mg/mL, from5.0 mg/mL to 9.0 mg/mL, from 6.0 mg/mL to 9.0 mg/mL, from 6.8 mg/mL to8.3 mg/mL, 6.9 mg/mL, 7.0 mg/mL, 7.1 mg/mL, 7.2 mg/mL, 7.3 mg/mL, 7.4mg/mL, 7.5 mg/mL, 7.6 mg/mL, 7.7 mg/mL, 7.8 mg/mL, 7.9 mg/mL, 8.0 mg/mL,8.1 mg/mL, 8.2 mg/mL or 8.3 mg/mL, or 6.8 mg/mL.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises protamine or protamine sulfate which ispresent in a concentration from 0.10, 0.15, 0.20, 0.25, 0.30, 0.32,0.35, 0.40, 0.45 or 0.5 mg/mL.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises a stabilizing agent, which is in oneembodiment a surfactant, a polyoxyethylene derivative of sorbitanmonolaurate (e.g. polysorbate 20), a polyethoxylethylene derivate ofoleic acid (e.g. polysorbate 80), poloxamer (which is apolyoxyethylene-polyoxypropylene copolymer), or polysorbate 20 orpolysorbate 80 or mixtures thereof. In another embodiment, thestabilizing agent, in one embodiment the surfactant, the polyoxyethylenederivative of sorbitan monolaurate (e.g. polysorbate 20), thepolyethoxylethylene derivate of oleic acid (e.g. polysorbate 80),poloxamer (which is a polyoxyethylene-polyoxypropylene copolymer), andin another polysorbate 20 or polysorbate 80 or mixtures thereof are/ispresent in a concentration from 0.01 to 0.05 mg/mL, 0.010 mg/mL, 0.015mg/mL, 0.020 mg/mL, 0.025 mg/mL, 0.03 mg/mL, or 0.02 mg/mL.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises more than one analogue and/or derivative ofinsulin, wherein one analogue and/or derivative of insulin is a fastacting insulin and one analogue and/or derivative of insulin is a longacting insulin. In another embodiment, the pharmaceutical formulationaccording to the present invention comprises a fast acting insulinselected from the group comprising insulin aspart, insulin lispro and/orinsulin glulisine and a long acting insulin selected from the groupcomprising insulin glargine, insulin detemir and/or insulin degludec.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises one or more further active pharmaceuticalingredients. In one embodiment the further active pharmaceuticalingredient is an antidiabetic agent. In another embodiment, thepharmaceutical formulation according to the present invention comprisesone or more antidiabetic agents as further active pharmaceuticalingredients selected from the group comprising: GLP-1 receptor agonists,dual GLP-1 receptor/glucagon receptor agonists, human FGF-21, FGF-21analogues, FGF-21 derivatives, insulins, human insulin, analogues ofinsulin, and derivatives of insulin. In another embodiment, thepharmaceutical formulation according to the present invention comprisesone or more further active pharmaceutical ingredients selected from thegroup comprising: insulin and insulin derivatives, GLP-1, GLP-1analogues and GLP-1 receptor agonists, polymer bound GLP-1 and GLP-1analogues, dual GLP1/GIP agonists, dual GLP1/Glucagon receptor agonists,PYY3-36 or analogues thereof, pancreatic polypeptide or analoguesthereof, glucagon receptor agonists or antagonists, GIP receptoragonists or antagonists, ghrelin antagonists or inverse agonists, Xeninand analogues thereof, DDP-IV inhibitors, SGLT2 inhibitors, dualSGLT2/SGLT1 inhibitors, biguanides thiazolidinediones, dual PPARagonists, sulfonylureas, meglitinides, alpha-glucosidase inhibitors,amylin and amylin analogues, GPR119 agonists, GPR40 agonists, GPR120agonists, GPR142 agonists, systemic or low-absorbable TGR5 agonists,Cycloset, inhibitors of 11-beta-HSD, activators of glucokinase,inhibitors of DGAT, inhibitors of protein tyrosinephosphatase 1,inhibitors of glucose-6-phosphatase, inhibitors offructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase,inhibitors of phosphoenol pyruvate carboxykinase, inhibitors of glycogensynthase kinase, inhibitors of pyruvate dehydrogenase kinase,alpha2-antagonists, CCR-2 antagonists, modulators of glucosetransporter-4, somatostatin receptor 3 agonists, HMG-CoA-reductaseinhibitors, fibrates, nicotinic acid and the derivatives thereof,nicotinic acid receptor 1 agonists, PPAR-alpha, gamma or alpha/gamma)agonists or modulators, PPAR-delta agonists, ACAT inhibitors,cholesterol absorption inhibitors, bile acid-binding substances, IBATinhibitors, MTP inhibitors, modulators of PCSK9, LDL receptorup-regulators by liver selective thyroid hormone receptor β agonists,HDL-raising compounds, lipid metabolism modulators, PLA2 inhibitors,ApoA-I enhancers, cholesterol synthesis inhibitors, lipid metabolismmodulators, omega-3 fatty acids and derivatives thereof, activesubstances for the treatment of obesity, such as sibutramine,tesofensine, orlistat, CB-1receptor antagonists, MCH-1 antagonists, MC4receptor agonists and partial agonists, NPY5 or NPY2 antagonists, NPY4agonists, beta-3-agonists, leptin or leptin mimetics, agonists of the5HT2c receptor, or the combinations of bupropione/naltrexone (CONTRAVE),bupropione/zonisamide (EMPATIC), bupropione/phentermine orpramlintide/metreleptin, QNEXA (Phentermine+topiramate), lipaseinhibitors, angiogenesis inhibitors, H3 antagonists, AgRP inhibitors,triple monoamine uptake inhibitors (norepinephrine and acetylcholine),MetAP2 inhibitors, nasal formulation of the calcium channel blockerdiltiazem, antisense oligonucleotides against production of fibroblastgrowth factor receptor 4, prohibitin targeting peptide-1, drugs forinfluencing high blood pressure, chronic heart failure oratherosclerosis, such as angiotensin II receptor antagonists, ACEinhibitors, ECE inhibitors, diuretics, beta-blockers, calciumantagonists, centrally acting hypertensives, antagonists of thealpha-2-adrenergic receptor, inhibitors of neutral endopeptidase,thrombocyte aggregation inhibitors.

In another embodiment, the pharmaceutical formulation according to thepresent invention comprises more than one analogue and/or derivative ofinsulin, wherein one analogue and/or derivative of insulin is a fastacting insulin and one analogue and/or derivative of insulin is a longacting insulin. In another embodiment the fast acting insulin isselected from the group comprising insulin aspart, insulin lispro and/orinsulin glulisine and the long acting insulin is selected from the groupcomprising insulin detemir and/or insulin degludec.

In another embodiment, the pharmaceutical formulation according to thepresent invention consists of (a). 3.496 mg/mL insulin aspart (rounded:3.5 mg/mL); and (b). 1.72 mg/mL metacresol; and (c). 1.50 mg/mL phenol;and (d). 0.0196 mg/mL Zn(II); and (e). 6.8 mg/mL sodium chloride; and(f). 0.02 mg/mL polysorbate 20; and (g). sodium hydroxide and/orhydrochloric acid to adjust the pH to 7.4 and (h). water.

In another embodiment, the pharmaceutical formulation according to thepresent invention consists of (a). 3.496 mg/mL insulin aspart (rounded:3.5 mg/mL); and (b). 1.72 mg/mL metacresol; and (c). 1.50 mg/mL phenol;and (d). 0.0196 mg/mL Zn(II); and (e). from 6.8 mg/mL to 8.3 mg/mLsodium chloride; (f). 0.02 mg/mL polysorbate 20; (g) from 0.1 mg/mL to0.5 mg/mL protamine sulfate; and (h). sodium hydroxide and/orhydrochloric acid to adjust the pH to a pH in the range from 7.1 to 7.6and (i). water.

In another embodiment, the pharmaceutical formulation according to thepresent invention consists of (a). 3.496 mg/mL insulin aspart (rounded:3.5 mg/mL); and (b). 1.72 mg/mL metacresol; and (c). 1.50 mg/mL phenol;and (d). 0.0196 mg/mL Zn(II); and (e). 6.8 or 6.9 or 7.0 or 7.1 or 7.2or 7.3 or 7.4 or 7.5 or 7.6 or 7.7 or 7.8 or 7.9 or 8.0 or 8.1 or 8.2 or8.3 mg/mL sodium chloride; (f). 0.02 mg/mL polysorbate 20; (g) 0.1 or0.15 or 0.2 or 0.25 or 0.3 or 0.32 or 0.35 or 0.4 or 0.45 or 0.5 mg/mLprotamine sulfate; and (h). sodium hydroxide and/or hydrochloric acid toadjust the pH to 7.4 and (i). water.

The present invention also provides a pharmaceutical formulation for usein the treatment of diabetes mellitus, hyperglycemia and/or for use inlowering blood glucose levels.

The present invention also provides a process for preparing thepharmaceutical formulation according to the present invention, whereinthe components are mixed together in the form of a solution orsuspension, the desired pH is adjusted and the mixture is made up to thefinal volume with water.

The present invention also relates to a kit or combination comprisingseparate packages of the pharmaceutical formulation according to thepresent invention and a medical device. In one embodiment the medicaldevice is selected from the group comprising: syringe, insulin injectionsystem, insulin infusion system, insulin pump, insulin pen injectiondevice.

The present invention also relates to a kit or combination comprisingseparate packages of the pharmaceutical formulation according to thepresent invention, of at least one further active pharmaceuticalingredient and optionally of a medical device. In one embodiment themedical device is selected from the group comprising: syringe, insulininjection system, insulin infusion system, insulin pump, insulin peninjection device.

The present invention also relates to a kit or combination comprisingseparate packages of the pharmaceutical formulation according to thepresent invention, of at least one further active pharmaceuticalingredient and optionally of a medical device, wherein the furtheractive pharmaceutical ingredient is an antidiabetic agent.

The present invention also relates to a kit or combination comprisingseparate packages of the pharmaceutical formulation according to thepresent invention, of at least one further active pharmaceuticalingredient and optionally of a medical device, wherein the furtheractive pharmaceutical ingredient is an antidiabetic agent selected fromthe group comprising: GLP-1 receptor agonists, dual GLP-1receptor/glucagon receptor agonists, human FGF-21, FGF-21 analogues,FGF-21 derivatives, insulins, human insulin, analogues of insulin, andderivatives of insulin. In another embodiment, the pharmaceuticalformulation according to the present invention comprises one or morefurther active pharmaceutical ingredients selected from the groupcomprising: insulin and insulin derivatives, GLP-1, GLP-1 analogues andGLP-1 receptor agonists, polymer bound GLP-1 and GLP-1 analogues, dualGLP1/GI P agonists, dual GLP1/Glucagon receptor agonists, PYY3-36 oranalogues thereof, pancreatic polypeptide or analogues thereof, glucagonreceptor agonists or antagonists, GIP receptor agonists or antagonists,ghrelin antagonists or inverse agonists, Xenin and analogues thereof,DDP-IV inhibitors, SGLT2 inhibitors, dual SGLT2/SGLT1 inhibitors,biguanides thiazolidinediones, dual PPAR agonists, sulfonylureas,meglitinides, alpha-glucosidase inhibitors, amylin and amylin analogues,GPR119 agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists,systemic or low-absorbable TGR5 agonists, Cycloset, inhibitors of11-beta-HSD, activators of glucokinase, inhibitors of DGAT, inhibitorsof protein tyrosinephosphatase 1, inhibitors of glucose-6-phosphatase,inhibitors of fructose-1,6-bisphosphatase, inhibitors of glycogenphosphorylase, inhibitors of phosphoenol pyruvate carboxykinase,inhibitors of glycogen synthase kinase, inhibitors of pyruvatedehydrogenase kinase, alpha2-antagonists, CCR-2 antagonists, modulatorsof glucose transporter-4, somatostatin receptor 3 agonists,HMG-CoA-reductase inhibitors, fibrates, nicotinic acid and thederivatives thereof, nicotinic acid receptor 1 agonists, PPAR-alpha,gamma or alpha/gamma) agonists or modulators, PPAR-delta agonists, ACATinhibitors, cholesterol absorption inhibitors, bile acid-bindingsubstances, IBAT inhibitors, MTP inhibitors, modulators of PCSK9, LDLreceptor up-regulators by liver selective thyroid hormone receptor βagonists, HDL-raising compounds, lipid metabolism modulators, PLA2inhibitors, ApoA-I enhancers, cholesterol synthesis inhibitors, lipidmetabolism modulators, omega-3 fatty acids and derivatives thereof,active substances for the treatment of obesity, such as sibutramine,tesofensine, orlistat, CB-1receptor antagonists, MCH-1 antagonists, MC4receptor agonists and partial agonists, NPY5 or NPY2 antagonists, NPY4agonists, beta-3-agonists, leptin or leptin mimetics, agonists of the5HT2c receptor, or the combinations of bupropione/naltrexone (CONTRAVE),bupropione/zonisamide (EMPATIC), bupropione/phentermine orpramlintide/metreleptin, QNEXA (Phentermine+topiramate), lipaseinhibitors, angiogenesis inhibitors, H3 antagonists, AgRP inhibitors,triple monoamine uptake inhibitors (norepinephrine and acetylcholine),MetAP2 inhibitors, nasal formulation of the calcium channel blockerdiltiazem, antisense oligonucleotides against production of fibroblastgrowth factor receptor 4, prohibitin targeting peptide-1, drugs forinfluencing high blood pressure, chronic heart failure oratherosclerosis, such as angiotensin II receptor antagonists, ACEinhibitors, ECE inhibitors, diuretics, beta-blockers, calciumantagonists, centrally acting hypertensives, antagonists of thealpha-2-adrenergic receptor, inhibitors of neutral endopeptidase,thrombocyte aggregation inhibitors.

The present invention also relates to a kit or combination comprisingseparate packages of the pharmaceutical formulation according to thepresent invention, of at least one further active pharmaceuticalingredient and optionally of a medical device, wherein the kit comprisesmore than one analogue and/or derivative of insulin, wherein oneanalogue and/or derivative of insulin is a fast acting insulin and oneanalogue and/or derivative of insulin is a long acting insulin. In oneembodiment the fast acting insulin is selected from the group comprisinginsulin aspart, insulin lispro and/or insulin glulisine and wherein thelong acting insulin is selected from the group comprising insulinglargine, insulin detemir and/or insulin degludec.

The present invention also relates to a kit or combination comprisingseparate packages of the pharmaceutical formulation according to thepresent invention, of at least one further active pharmaceuticalingredient and optionally of a medical device for use in the treatmentof diabetes mellitus, hyperglycemia and/or for use in lowering bloodglucose levels.

In another embodiment, the present invention also relates to a kit orcombination comprising separate packages of the pharmaceuticalformulation according to the present invention, of at least one furtheractive pharmaceutical ingredient and optionally of a medical device,wherein the pharmaceutical formulation according to the presentinvention and the further active pharmaceutical ingredient, in oneembodiment an antidiabetic agent, are administered continuously,separately, sequentially and/or stepwise.

The present invention also relates to the use of a medical device foradministering the pharmaceutical formulation to an animal and/or human.In one embodiment, the medical device is selected from the groupcomprising: syringe, insulin injection system, insulin infusion system,insulin pump, insulin pen injection device

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows liquid chromatography-mass spectrometry (LC/MS) of TRISbuffered formulation (formulation no._0104).

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to a fill material containing “a carrier” includesone or more carriers, reference to “an additive” includes reference toone or more of such additives.

As used herein, the term “active pharmaceutical ingredient” (API)includes any pharmaceutically active chemical or biological compound andany pharmaceutically acceptable salt thereof and any mixture thereof,that provides some pharmacologic effect and is used for treating orpreventing a condition. Exemplary pharmaceutically acceptable saltsinclude hydrochloric, sulfuric, nitric, phosphoric, hydrobromic,maleric, malic, ascorbic, citric, tartaric, pamoic, lauric, stearic,palmitic, oleic, myristic, lauryl sulfuric, naphthalinesulfonic,linoleic, linolenic acid, and the like. As used herein, the terms“active pharmaceutical ingredient”, “drug”, “active agent”, “activeingredient”, “active substance” and “drug” are meant to be synonyms,i.e., have identical meaning.

In a one embodiment the active pharmaceutical ingredient is anantidiabetic agent. Examples of antidiabetic agents are found in theRote Liste 2012, chapter 12. Examples of antidiabetic agents include butnot limited to (a) insulin, insulin analogues and insulin derivatives,(b) glucagon-like-peptide 1 (GLP-1) and its analogues and receptoragonists, (c) dual GLP-1/GIP agonists, and (d) dual GLP-1/glucagonreceptor agonists, as described in detail next.

(a). Insulin, insulin analogues, and insulin derivatives

Examples of insulin, insulin analogues, and insulin derivatives includebut are not limited to insulin glargine (Lantus®), insulin glulisine(Apidra®), insulin detemir (Levemir®), insulin lispro(Humalog®/Liprolog®), insulin degludec (Tresiba®), insulin aspart(NovoLog®/NovoRapid®), basal insulin and analogues (e.g. LY2605541,LY2963016), PEGylated insulin lispro, Humulin®, Linjeta®, SuliXen®,NN1045, insulin plus Symlin®, fast-acting and short-acting insulins(e.g. Linjeta®, PH20 insulin, NN1218, HinsBet®), oral, inhalable,transdermal and sublingual insulins (e.g. Exubera®, Nasulin®, Afrezza®,insulin tregopil, TPM-02/Insulin, Capsulin®, Oral-lyn®, Cobalamin® oralinsulin, ORMD-0801, NN1953, VlAtab®). Additionally included are alsothose insulin derivatives which are bonded to albumin or another proteinby a bifunctional linker.

(b). Glucagon-like-peptide 1 (GLP-1), GLP-1 analogues and GLP-1 receptoragonists

Examples of GLP-1, GLP-1 analogues and GLP-1 receptor agonists includebut are not limited to lixisenatide (AVE0010/ZP10/Lyxumia®),exenatide/exendin-4 (Byetta®/Bydureon®/ITCA 650, liraglutide/Victoza®),semaglutide, taspoglutide, albiglutide, dulaglutide, rExendin-4,CJC-1134-PC, PB-1023, TTP-054, HM-11260C, CM-3, GLP-1 Eligen, ORMD-0901,NN9924, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, MAR-701, ZP-2929,ZP-3022, CAM-2036, DA-15864, ARI-2651, ARI-2255, exenatide-XTEN andglucagon-XTEN, AMX-8089+VRS-859 and polymer bound GLP-1 and GLP-1analogues.

(c). Dual GLP-1/glucose-dependent insulinotropic peptides (GIP) agonists

Examples of dual GLP-1/GIP agonists include but are not limited toMAR701, MAR-709, BHM081/BHM089/BHM098).

(d). Dual GLP-1/glucagon receptor agonists

Examples of dual GLP-1/glucagon receptor agonists include but are notlimited to OAP-189 (PF-05212389, TKS-1225), TT-401/402, ZP2929,LAPS-HMOXM25, MOD-6030).

Other suitable active pharmaceutical ingredients which may be includedin the pharmaceutical formulations of the invention include but are notlimited to the following:

Further gastrointestinal peptides such as peptide YY 3-36 (PYY3-36) oranalogues thereof and pancreatic polypeptide (PP) or analogues thereof.

Glucagon receptor agonists or antagonists, GIP receptor agonists orantagonists, ghrelin antagonists or inverse agonists and xenin andanalogues thereof.

Dipeptidyl peptidase-IV (DPP-4) inhibitors, for example:alogliptin/Nesina®, linagliptin/BI-1356/Ondero®/Trajenta®/ Tradjenta®/Trayenta®, saxagliptin/Onglyza®, sitagliptin/Januvia®/Xelevia®/Tesavel®,sitagliptin+metformin/Janumet®/ Velmetia®, aildagliptin, anagliptin,aemigliptin, tenegliptin, melogliptin, trelagliptin, DA-1229, MK-3102,KM-223, KRP-104 and Ari-2243.

Sodium-dependent glucose transporter 2 (SGLT2) inhibitors, for example:canagliflozin, dapagliflozin, remogliflozin, sergliflozin,empagliflozin, ipragliflozin, tofogliflozin (RO-4998452),luseogliflozin, LX-4211, ertugliflozin (PF-04971729), EGT-0001442 andDSP-3235.

Dual SGLT2/SGLT1 inhibitors.

Biguanides (e.g. metformin, buformin, phenformin), thiazolidinediones(e.g. pioglitazone, rivoglitazone, rosiglitazone, troglitazone), dualPPAR agonists (e.g. aleglitazar, muraglitazar, tesaglitazar),sulfonylureas (e.g. tolbutamide, glibenclamide, glimepiride/Amaryl®,glipizide), meglitinides (e.g. nateglinide, repaglinide, mitiglinide),alpha-glucosidase inhibitors (e.g. acarbose, miglitol, voglibose),amylin and amylin analogues (e.g. pramlintide/Symlin®).

G-protein coupled receptor 119 (GPR119) agonists (e.g. GSK-1292263,PSN-821, MBX-2982, APD-597, ARRY-981).

GPR40 agonists (e.g. TAK-875, TUG-424, P-1736, JTT-851, GW9508).

GPR120 agonists and GPR142 agonists.

Systemic or low-absorbable TGR5 (GPBAR1=G-protein-coupled bile acidreceptor 1) agonists (e.g. INT-777, XL-475, SB756050).

Bromocriptine/Cycloset®, inhibitors of 11-beta-hydroxysteroiddehydrogenase (11-beta-HSD) (e.g. LY2523199, BMS770767, RG-4929,BMS816336, AZD-8329, HSD-016, BI-135585), activators of glucokinase(e.g. PF-04991532, TTP-399, GK1-399, ARRY-403 (AMG-151), TAK-329,ZYGK1), inhibitors of diacylglycerol O-acyltransferase (DGAT) (e.g.pradigastat (LCQ-908), LCQ-908), inhibitors of proteintyrosinephosphatase 1 (e.g. trodusquemine), inhibitors of glucosephosphatase, inhibitors of fructose-1,6-bisphosphatase, inhibitors ofglycogen phosphorylase, inhibitors of phosphoenol pyruvatecarboxykinase, inhibitors of glycogen synthase kinase, inhibitors ofpyruvate dehydrogenase kinase, alpha2 adrenergic receptor antagonists,C-C chemokine receptor type 2 (CCR-2) antagonists, modulators of glucosetransporter-4 and somatostatin receptor 3 agonists (e.g. MK-4256).

One or more lipid lowering agents are also suitable as activepharmaceutical ingredients, such as for example:3-hydroxy-3-methylglutaryl-coenzym-A-reductase (HMG-CoA-reductase)inhibitors (e.g. simvastatin, atorvastatin, rosuvastatin), fibrates(e.g. bezafibrate, fenofibrate), nicotinic acid and derivatives thereof(e.g. niacin, including slow release formulations of niacin), nicotinicacid receptor 1 agonists (e.g. GSK-256073), peroxisomeproliferator-activated receptors (PPAR-)(alpha, gamma or alpha/gamma)agonists or modulators (e.g. aleglitazar), PPAR-delta agonists,acetyl-CoA-acetyltransferase (ACAT) inhibitors (e.g. avasimibe),cholesterol absorption inhibitors (e.g. ezetimibe), bile acid-bindingsubstances (e.g. cholestyramine, colesevelam), ileal bile acid transportinhibitors (IBAT) (e.g. GSK-2330672), microsomal triglyceride transferprotein (MTP) inhibitors (e.g. lomitapide (AEGR-733), SLx-4090,granotapide), modulators of proprotein convertase subtilisin/kexin type9 (PCSK9) (e.g. REGN727/SAR236553, AMG-145, LGT-209, PF-04950615,MPSK3169A, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001,ISIS-394814, 1B20, LGT-210, 1D05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), LDLreceptor up-regulators, for example liver selective thyroid hormonereceptor beta agonists (e.g. eprotirome (KB-2115), MB07811, sobetirome(QRX-431), VIA-3196, ZYT1), HDL-raising compounds such as: CETPinhibitors (e.g. torcetrapib, anacetrapib (MK0859), dalcetrapib,evacetrapib, JTT-302, DRL-17822, TA-8995, R-1658, LY-2484595) or ABC1regulators, lipid metabolism modulators (e.g. BMS-823778, TAP-301,DRL-21994, DRL-21995), phospholipase A2 (PLA2) inhibitors (e.g.darapladib/Tyrisa®, varespladib, rilapladib), ApoA-I enhancers (e.g.RVX-208, CER-001, MDCO-216, CSL-112, VRX-HDL, VRX-1243, VIRxSYS),cholesterol synthesis inhibitors (e.g. ETC-1002) and lipid metabolismmodulators (e.g. BMS-823778, TAP-301, DRL-21994, DRL-21995) and omega-3fatty acids and derivatives thereof (e.g. icosapent ethyl (AMR101),Epanova®, AKR-063, NKPL-66).

Other suitable active pharmaceutical ingredients which may be includedin the pharmaceutical formulations include one or more active substancesfor the treatment of obesity, including but not limited to:

Sibutramine, tesofensine, orlistat, cannabinoid receptor 1 (CB1)antagonists (e.g. TM-38837), melanin-concentrating hormone (MCH-1)antagonists (e.g. BMS-830216, ALB-127158(a)), MC4 receptor agonists andpartial agonists (e.g. AZD-2820, RM-493), neuropeptide Y5 (NPY5) or NPY2antagonists (e.g. velneperit, S-234462), NPY4 agonists (e.g. PP-1420),beta adrenergic receptor agonists, leptin or leptin mimetics, agonistsof the 5-hydroxytryptamine 2c (5HT2c) receptor (e.g. lorcaserin), or thecombinations of bupropione/naltrexone (Contrave®), bupropione/zonisamide(Empatic®), bupropione/phentermine or pramlintide/metreleptin,phentermine/topiramate (Qsymia®), lipase inhibitors (e.g.cetilistat/Cametor®), angiogenesis inhibitors (e.g. ALS-L1023),histamine H3 antagonists (e.g. HPP-404), AgRP (agouti related protein)inhibitors (e.g. TTP-435), triple monoamine uptake inhibitors (dopamine,norepinephrine and serotonin reuptake) (e.g. tesofensine), methionineaminopeptidase 2 (MetAP2) inhibitors (e.g. beloranib), nasalformulations of the calcium channel blocker diltiazem (e.g. CP-404) andantisense oligonucleotides against production of fibroblast growthfactor receptor 4 (FGFR4) (e.g. ISIS-FGFR4Rx) or prohibitin targetingpeptide-1 (e.g. Adipotide®).

Further suitable active pharmaceutical ingredients which may be includedin the pharmaceutical formulations include but are not limited to:

Angiotensin II receptor antagonists (e.g. telmisartan, candesartan,valsartan, losartan, eprosartan, irbesartan, olmesartan, tasosartan,azilsartan), angiotensin converting enzyme (ACE) inhibitors, endothelinconverting enzyme (ECE) inhibitors, diuretics, beta-blockers, calciumantagonists, centrally acting hypertensives, antagonists of thealpha-2-adrenergic receptor, inhibitors of neutral endopeptidase,thrombocyte aggregation inhibitors and others or combinations thereofare suitable.

As used herein, the terms “analogue of insulin” and “insulin analogue”refer to a polypeptide which has a molecular structure which formallycan be derived from the structure of a naturally occurring insulin, forexample that of human insulin, by deleting and/or exchanging at leastone amino acid residue occurring in the naturally occurring insulinand/or adding at least one amino acid residue. The added and/orexchanged amino acid residue can either be codable amino acid residuesor other naturally occurring residues or purely synthetic amino acidresidues. Examples of analogues of insulin include, but are not limitedto, the following:

(i). ‘Insulin aspart’ is created through recombinant DNA technology sothat the amino acid B28 in human insulin (i.e. the amino acid no. 28 inthe B chain of human insulin), which is proline, is replaced by asparticacid;

(ii). ‘Insulin lispro’ is created through recombinant DNA technology sothat the penultimate lysine and proline residues on the C-terminal endof the B-chain of human insulin are reversed (human insulin:Pro^(B28)Lys^(B29); insulin lispro: Lys^(B28)Pro^(B29));

(iii). ‘Insulin glulisine’ differs from human insulin in that the aminoacid asparagine at position B3 is replaced by lysine and the lysine inposition B29 is replaced by glutamic acid;

(iv). “Insulin glargine” differs from human insulin in that theasparagine at position A21 is replaced by glycine and the B chain isextended at the carboxy terminal by two arginines.

As used herein, the term “aqueous” refers to a solution in which thesolvent is water and/or to a suspension in which the external phase iswater and/or to an emulsion in which the dispersed or continuous phaseis water.

As used herein, the term “buffering agent” refers to a weak acid or baseused to maintain the acidity (pH) of a solution, a suspension and/or anemulsion near a chosen value after the addition of another acid or base.The function of a buffering agent is to prevent a rapid change in the pHvalue when acids or bases are added to the solution. In an aqueoussolution, suspension and/or emulsion, a buffering agent is present in amixture of a weak acid and its conjugate base or a in a mixture of aweak base and its conjugated acid. Examples of buffering agents include,but are not limited to, the following: sodium bicarbonate; acetic acidor acetate salts (e.g. sodium acetate, zinc acetate); boric acid orboric salts; N-cyclohexyl-2-aminoethanesulfonic acid (CHES) or saltsthereof;3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid (TAPS) or salts thereof; 2-(N-morpholino)ethanesulfonic acid (MES)and salts thereof; piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES) andsalts thereof; N-(2-acetamido)-2-aminoethane-sulfonic acid (ACES) andsalts thereof; cholamine chloride; BES;2-[[1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl]amino]ethanesulfonicacid (TES) and salts thereof;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) andsalts thereof; acetamidoglycine;N-(2-hydroxy-1,1-bis(hydroxyl-methyl)ethyl)glycine (tricine);glycinamide; 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine) and saltsthereof; propionate salts;3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]-amino]-2-hydroxy-propane-1-sulfonicacid (TAPSO) and salts thereof; 3-morpholinopropane-1-sulfonic acid(MOPS) and salts thereof; saline-sodium citrate (SSC) buffer;2-amino-2-hydroxymethyl-propane-1,3-diol (synonyms: TRIS, trisamine,THAM, tromethamine, trometamol, tromethane); citric acid or citratesalts (e.g. sodium citrate); trisodium phosphate, disodium hydrogenphosphate, sodium dihydrogen phosphate, tripotassium phosphate,dipotassium phosphate, monopotassium phosphate and/or any otherbuffering agent containing phosphate.

Amino acids (having free basic or acidic functional groups, e.g.methionin, arginine) or peptides (having free basic or acidic functionalgroups) may also be used as buffering agent. As used herein, the term“buffering agent” also comprises amino acids, peptides and proteins. Asinsulin analogues and/or insulin derivatives and/or protamine arepeptides or derivatives of peptides (i.e. both contain amino acidshaving free basic or acidic functional groups), they may also have acertain buffering capacity, i.e. are also to be considered as bufferingagent.

As used herein, the term “fast acting insulin” or “short acting insulin”refers to insulin analogues and/or insulin derivatives, wherein theinsulin-mediated effect begins within 5 to 15 minutes and continues tobe active for 3 to 4 hours. Examples of fast acting insulins include,but are not limited to, the following: (i). insulin aspart; (ii).insulin lispro and (iii). insulin glulisine.

As used herein, the terms “free of additional buffering agent” or“buffer-free” means that there is no further buffering agent next to theanalogue and/or derivative of insulin and optionally protamine. Asmentioned above, analogues and/or derivatives of insulin as well asprotamine contain amino acids having acidic or basic side chains and aretherefore also to be considered as buffering agent. Independently fromthe absence of any buffering agents, the aqueous pharmaceuticalformulation according to the present invention may optionally compriseprotamine.

As used throughout the description and the claims of this specification,the word “comprise” and variations of the word, such as “comprising” and“comprises” is not intended to exclude other additives, components,integers or steps.

As used herein, the terms “derivative of insulin” and “insulinderivative” refer to a polypeptide which has a molecular structure whichformally can be derived from the structure of a naturally occurringinsulin, for example that of human insulin, in which one or more organicsubstituents (e.g. a fatty acid) is bound to one or more of the aminoacids. Optionally, one or more amino acids occurring in the naturallyoccurring insulin may have been deleted and/or replaced by other aminoacids, including non-codeable amino acids, or amino acids, includingnon-codable, have been added to the naturally occurring insulin.Examples of derivatives of insulin include, but are not limited to, thefollowing:

(i). ‘Insulin detemir’ which differs from human insulin in that theC-terminal threonine in position B30 is removed and a fatty acid residue(myristic acid) is attached to the epsilon-amino function of the lysinein position B29.

(ii). ‘Insulin degludec’ which differs from human insulin in that thelast amino acid is deleted from the B-chain and by the addition of aglutamyl link from LysB29 to a hexadecandioic acid.

As used herein, the term “FGF-21” means “fibroblast growth factor 21”.FGF-21 compounds may be human FGF-21, an analogue of FGF-21 (referred to“FGF-21 analogue”) or a derivative of FGF-21 (referred to “FGF-21derivative”).

As used herein, the term “formulation” refers to a product comprisingspecified ingredients in predetermined amounts or proportions, as wellas any product that results, directly or indirectly, from combiningspecified ingredients in specified amounts. In relation topharmaceutical formulations, this term encompasses a product comprisingone or more active ingredients, and an optional carrier comprising inertingredients, as well as any product that results, directly orindirectly, from combination, complexation or aggregation of any two ormore of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. In general, pharmaceutical formulations areprepared by uniformly bringing the active pharmaceutical ingredient(i.e. the analogue and/or derivative of insulin) into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation. Thepharmaceutical formulation includes enough of the active pharmaceuticalingredient to produce the desired effect upon the progress or conditionof diseases. As used herein, the term “formulation” may refer to asolution as well as to a suspension or to an emulsion. As used herein,the terms “formulation” and “composition” are meant to be synonyms,i.e., have identical meaning. The pharmaceutical compositions are madefollowing conventional techniques of pharmaceutical technology involvingmixing, filling and dissolving the ingredients, as appropriate, to givethe desired oral, parenteral, rectal, transdermal, or topical products.

As used herein, the term “GLP-1 receptor agonist” refers to compoundswhich have an agonistic activity at the glucagon-like peptide-1receptor. Examples of GLP-1 receptor agonists include, but are notlimited to, the following: exenatide/exendin-4, liraglutide,lixisenatide, dulaglutide, albiglutide, semaglutide, taspoglutide,rExendin-4, CJC-1134-PC, PB-1023, TTP-054, HM-11260C, CM-3, GLP-1Eligen, ORMD-0901, NN9924, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1,ZYD-1, MAR-701, ZP-2929, ZP-3022, CAM-2036, DA-15864, ARI-2651,ARI-2255, exenatide-XTEN and glucagon-XTEN, AMX-8089+VRS-859 and polymerbound GLP-1 and GLP-1 analogues.

As used herein, the term “dual GLP-1 receptor/glucagon receptor agonist”refers to compounds which have agonistic activity at both the GLP-1receptor and the glucacon receptor. Examples of dual GLP-1receptor/glucagon receptor agonist include, but are not limited to, thefollowing: oxyntomodulin, MAR701, MAR-709, and BHM081/BHM089/BHM098.

As used herein, the term “human insulin” refers to the human hormonewhose structure and properties are well-known. Human insulin has twopolypeptide chains (chains A and B) that are connected by disulphidebridges between cysteine residues, namely the A-chain and the B-chain.

The A-chain is a 21 amino acid peptide and the B-chain is a 30 aminoacid peptide, the two chains being connected by three disulphidebridges: one between the cysteins in position 6 and 11 of the A-chain;the second between the cysteine in position 7 of the A-chain and thecysteine in position 7 of the B-chain; and the third between thecysteine in position 20 of the A-chain and the cysteine in position 19of the B-chain.

As used herein, the term “including” is used to mean “including but notlimited to”. “Including” and “including but not limited to” are usedinterchangeably.

As used herein, the term “isoelectric point” (pI, IEP) refers to the pHvalue at which a particular molecule carries no net electrical charge.The isoelectric point can be determined by using isoelectric focusing,which is a technique for separating different molecules by differencesin their isoelectric point and which is well known in the art. It canalso be calculated (see e.g. Levene and Simms, ‘Calculation ofisoelectric point’ J. Biol. Chem., 1923, pp. 801-813).

As used herein, the term “kit” refers to a product (e.g. medicament,kit-of-parts) comprising one package or one or more separate packagesof:

(i). A pharmaceutical formulation containing an active pharmaceuticalingredient and at least one further active pharmaceutical ingredient andoptionally a medical device. The at least one further activepharmaceutical ingredient may be present in said pharmaceuticalformulation, i.e. the kit may comprise one or more packages, whereineach package comprises one pharmaceutical formulation which comprisestwo or more active pharmaceutical ingredients. The further activepharmaceutical ingredient may also be present in a furtherpharmaceutical formulation, i.e. the kit may comprise separate packagesof two or more pharmaceutical formulations, wherein each pharmaceuticalformulation contain one active pharmaceutical ingredient.

Or

(ii). A pharmaceutical formulation containing an active pharmaceuticalingredient and medical device.

A kit may comprise one package only or may comprise one or more separatepackages For example, the kit may be a product (e.g. medicament)containing two or more vials each containing a defined pharmaceuticalformulation, wherein each pharmaceutical formulation contains at leastone active pharmaceutical ingredient. For example, the kit may comprise(i.) a vial containing a defined pharmaceutical formulation and (ii).further a tablet, capsule, powder or any other oral dosage form whichcontains at least one further active pharmaceutical ingredient.

The kit may further comprise a package leaflet with instructions for howto administer the pharmaceutical formulation and the at least onefurther active pharmaceutical ingredient.

As used herein, the term “medical device” means any instrument,apparatus, implant, in vitro reagent or similar or related article thatis used to diagnose, prevent, or treat a disease of other condition, anddoes not achieve its purpose through pharmacological action within or onthe body. As used herein, a medical device may be a syringe, an insulininjection system, an insulin infusion system, an insulin pump or aninsulin pen injection device. As used herein, a medical device may bemechanically or electromechanically driven.

As used herein, unless specifically indicated otherwise, the conjunction“or” is used in the inclusive sense of “and/or” and not the exclusivesense of “either/or”.

As used herein, the term “pH” and “pH value” refer to the decimallogarithm of the reciprocal of the hydrogen ion activity in a solution.

As used herein, the term “pharmaceutical” refers to the intended use inthe medical diagnosis, cure, treatment and/or prevention of diseases.

As used herein, the term “pharmaceutically acceptable” refers tophysiologically well tolerated by a mammal or a human.

As used herein, the term “protamine” refers to a mixture of stronglybasic peptides. It was originally isolated from the sperm of salmon andother species of fish but is now produced primarily recombinant throughbiotechnology. It contains more than two-thirds of L-arginine. Asprotamine contains amino acids having free basic side chains, it has acertain buffering capacity and is therefore considered to be a bufferingagent. Protamine may be used as protamine sulfate or protaminehydrochloride.

Concentrations, amounts, solubilities, particle size, wavelength, pHvalues, weight mass, molecular weight, percent and other numerical datemay be expressed or presented herein in a range format. It is to beunderstood that such a range format is used merely for convenience andbrevity and thus should be interpreted flexibly to include not only thenumerical values explicitly recited as the limits of the range, but alsoto include all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited.

As used herein, the term “long acting insulin” refers to insulinanalogues and/or insulin derivatives, wherein the insulin-mediatedeffect begins within 0.5 to 2 hours and continues to be active for aboutor more than 24 hours. Examples of fast acting insulins include, but arenot limited to, the following: (i). insulin glargine; (ii). insulinedetemir and (iii). insulin degludec.

As used herein, the term “stability” refers to the chemical and/orphysical stability of active pharmaceutical ingredients, in particularof insulin analogues and/or derivatives. The purpose of stabilitytesting is to provide evidence on how the quality of an activepharmaceutical ingredient or dosage form varies with time under theinfluence of a variety of environmental factors such as temperature,humidity, and light, and to establish a shelf life for the activepharmaceutical ingredient or dosage form and recommended storageconditions. Stability studies can include testing of those attributes ofthe active pharmaceutical ingredient that are susceptible to changeduring storage and are likely to influence quality, safety, and/orefficacy. The testing can cover, as appropriate, the physical, chemical,biological, and microbiological attributes, preservative content (e.g.,antioxidant, antimicrobial preservative), and functionality tests (e.g.for a dose delivery system). Analytical procedures can be fullyvalidated and stability indicating. In general, significant changes foran active pharmaceutical ingredient and/or dosage form with regard tostability are defined as:

-   -   a 5% change in assay from its initial value; or failure to meet        the acceptance criteria for potency when using biological or        immunological procedures;    -   any degradation products exceeding its acceptance criterion;    -   failure to meet the acceptance criteria for appearance, physical        attributes, and functionality test (e.g., color, phase,        separation, resuspendibility, caking, hardness, dose delivery        per actuation); however, some changes in physical attributes        (e.g. softening of suppositories, melting of creams) may be        expected under accelerated conditions;    -   and, as appropriate for the dosage form:    -   failure to meet the acceptance criterion for pH; or    -   failure to meet the acceptance criteria for dissolution for 12        dosage units.

The significant changes may also be evaluated against establishedacceptance criteria prior to starting the evaluation of the stability.

Acceptance criteria can be derived from the monographs (e.g. monographsfor the European Pharmacopeia, of the United States Pharmacopeia, of theBritish Pharmacopeia, or others), and from the analytical batches of theactive pharmaceutical ingredient and medicinal product used in thepreclinical and clinical studies. Acceptable limits should be proposedand justified, taking into account the levels observed in material usedin preclinical and clinical studies. Product characteristics may bevisual appearance, purity, color and clarity for solutions/suspensions,visible particulates in solutions, and pH. As a non-limiting example,suitable acceptance criteria for insulin aspart formulations are shownbelow:

Test item Acceptance criteria for clinical trials Appearance of solution(visual) Clarity and degree of opalescence Monitoring Degree ofcoloration Monitoring Assay insulin aspart units (HPLC) 90.0 insulinaspart units/mL to 110.0 insulin aspart units/mL Related impurities(HPLC) B28isoAsp insulin aspart equal or below to 2.5 % Total of A21Aspinsulin aspart, equal or below to 5.0 % B3Asp insulin aspart andB3isoAsp insulin aspart Any other unspecified, equal or below to 2.0 %unidentified impurity Total of other impurities equal or below to 3.5 %High molecular weight proteins equal or below to 1.5 % (HPSEC) PH 7.0 to7.8 Particulate matter (visible particles) Practically free of visibleparticles Particulate matter (subvisible Number of particles percontainer: particles) equal or larger to 10 μm: equal or below to 6000equal or larger to 25 μm: equal or below to 600 Assay m-cresol 1.55 to1.89 [mg/mL] Assay phenol 1.35 to 1.65 [mg/mL] Zinc (Zn(II)) (AAS) below40 μg per 100 units insulin aspart

The acceptance criteria shown above are based on monographed acceptancelimits (e.g. British Pharmacopoeia, Volume III, 2012 or PharmacopoeialForum, Volume 36(6), November-December 2010) and/or are derived fromextensive experience in the development of insulin formulations.

As used herein, the term “treatment” refers to any treatment of amammalian, for example human condition or disease, and includes: (1)inhibiting the disease or condition, i.e., arresting its development,(2) relieving the disease or condition, i.e., causing the condition toregress, or (3) stopping the symptoms of the disease.

As used herein, the unit of measurement “U” and/or “international units”refers to the blood glucose lowering activity of insulin and is defined(according to the World Health Organization, WHO) as follows: 1 Ucorresponds to the amount of highly purified insulin (as defined by theWHO) which is sufficient to lower the blood glucose level of a rabbit(having a body weight of 2-2.5 kg) to 50 mg/100 mL within 1 hour and to40 mg/100 mL within 2 hours. For human insulin, 1 U corresponds toapproximately 35 μg (Lill, Pharmazie in unserer Zeit, No. 1, pp. 56-61,2001). For insulin aspart, 100 U correspond to 3.5 mg (productinformation NovoRapid®). For insulin lispro, 100 U correspond to 3.47 mg(product information Humalog®). For insulin glulisine, 100 U correspondto 3.49 mg (product information Apidra® cartridges). For insulindetermir, 100 U correspond to 14.2 mg (product information Levemir®).For insulin glargine, 100 U correspond to 3.64 mg (product informationLantus®).

Further embodiments of the present invention include the following:

In one aspect, the invention provides a pharmaceutical formulationcomprising (a). at least one analogue and/or derivative of insulin; and(b). Zn(II); and (c). sodium chloride; and (d). optionally protamine;wherein the pharmaceutical formulation has a pH value in the range offrom 6.0 to 9.0 and is free of any additional buffering agent.

In one aspect, the pharmaceutical formulation of the invention is anaqueous pharmaceutical formulation.

In one aspect, the pharmaceutical formulation of the invention has a pHvalue in the range from 7.0 to 7.8.

In one aspect, the pharmaceutical formulation of the invention comprisesan analogue of insulin selected from the group consisting of insulinaspart, insulin lispro and insulin glulisine.

In one aspect, the pharmaceutical formulation of the invention comprisesa derivative of insulin which is insulin detemir and/or insulindegludec.

In one aspect, the pharmaceutical formulation of the invention comprisesan analogue and/or derivative of insulin which is present in aconcentration from 10 U/mL to 1000 U/mL.

In one aspect, the pharmaceutical formulation of the invention comprisesZn(II) in a concentration from 0.0100 to 0.0600 mg/100 U of the analogueand/or derivative of insulin.

In one aspect, the pharmaceutical formulation of the invention comprisessodium chloride in a concentration from 0.01 to 15 mg/mL.

In one aspect, the pharmaceutical formulation of the invention comprisessodium chloride in a concentration from 6.8 to 8.3 mg/mL.

In one aspect, the pharmaceutical formulation of the invention comprisesprotamine sulfate which in a concentration from 0.1 to 0.5 mg/mL.

In one aspect, the pharmaceutical formulation of the invention is freeof any additional buffering agent selected from the group consisting of2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), phosphate, citric acid,citrate, acetic acid, acetate, glycylglycine and methionine.

In one aspect, the pharmaceutical formulation of the invention comprisesone or more further active pharmaceutical ingredients.

In one aspect, the pharmaceutical formulation of the invention comprisesa further active pharmaceutical ingredient which is an antidiabeticagent.

In one aspect, the pharmaceutical formulation of the invention comprisesa further active pharmaceutical ingredient which is an antidiabeticagent selected from the group consisting of (a) a GLP-1 receptoragonist; (b) a dual GLP-1 receptor/glucagon receptor agonist; (c) humanFGF-21; (d) an FGF-21 analogue; (e) an FGF-21 derivative; (f) insulin;(g) human insulin; (h) an analogue of insulin; and (i) a derivative ofinsulin.

In one aspect, the pharmaceutical formulation of the invention comprisesmore than one analogue and/or derivative of insulin, wherein oneanalogue and/or derivative of insulin is a fast acting insulin and oneanalogue and/or derivative of insulin is a long acting insulin.

In one aspect, the pharmaceutical formulation of the invention comprisesa fast acting insulin is selected from the group consisting of insulinaspart, insulin lispro, and insulin glulisine, and wherein the longacting insulin is one or more insulin selected from the group consistingof insulin detemir and insulin degludec.

In one aspect, the pharmaceutical formulation of the invention consistsof:

-   -   (a). 3.5 mg/mL insulin aspart;    -   (b). 1.72 mg/mL metacresol;    -   (c). 1.50 mg/mL phenol;    -   (d). 0.0196 mg/mL Zn(II);    -   (e). 6.8 mg/mL sodium chloride;    -   (f). 0.02 mg/mL polysorbate 20;    -   (g). sodium hydroxide and/or hydrochloric acid to adjust the pH        to 7.4, and    -   (h). water.

In one aspect, the pharmaceutical formulation of the invention consistsof:

-   -   (a). 3.5 mg/mL insulin aspart;    -   (b). 1.72 mg/mL metacresol;    -   (c). 1.50 mg/mL phenol;    -   (d). 0.0196 mg/mL Zn(II);    -   (e). from 6.8 mg/mL to 8.3 mg/mL sodium chloride;    -   (f). 0.02 mg/mL polysorbate 20;    -   (g). from 0.1 mg/mL to 0.5 mg/mL protamine sulfate;    -   (h). sodium hydroxide and/or hydrochloric acid to adjust the pH        to a pH in the range from 7.1 to 7.6; and    -   (i). water.

In one aspect, the invention provides a process for preparing thepharmaceutical formulation of the invention, wherein the components aremixed together in the form of a solution or suspension, the pH isadjusted to reach the desired pH, and water is added to reach the finalvolume.

In one aspect, the invention provides a kit comprising one or moreseparate packages of:

-   -   (a). a pharmaceutical formulation of the invention; and    -   (b). a medical device.

In one aspect, the invention provides a kit comprising one or moreseparate packages of:

-   -   (a). a pharmaceutical formulation of the invention; and    -   (b). at least one further active pharmaceutical ingredient;    -   (c). and optionally a medical device.

In one aspect, the kit of the invention comprises a further activepharmaceutical ingredient which is an antidiabetic agent.

In one aspect, the kit of the invention comprises a further activepharmaceutical ingredient which is an antidiabetic agent selected fromthe group consisting of:

-   -   (a). a GLP-1 receptor agonist;    -   (b). a dual GLP-1 receptor/glucagon receptor agonist;    -   (c). human FGF-21;    -   (d). an FGF-21 analogue;    -   (e). an FGF-21 derivative;    -   (f). insulin;    -   (g). human insulin;    -   (h). an analogue of insulin; and    -   (i). a derivative of insulin.

In one aspect, the kit of the invention comprises more than one analogueand/or derivative of insulin, wherein one analogue and/or derivative ofinsulin is a fast acting insulin and one analogue and/or derivative ofinsulin is a long acting insulin.

In one aspect, the kit of the invention comprises a fast acting insulinselected from the group consisting of insulin aspart, insulin lispro andinsulin glulisine, and a long acting insulin selected from the groupconsisting of insulin glargine, insulin detemir and insulin degludec.

In one aspect, the invention provides a pharmaceutical formulation orkit for use in the treatment of diabetes mellitus.

In one aspect, the invention provides a pharmaceutical formulation orkit for use in the treatment of hyperglycemia.

In one aspect, the invention provides a pharmaceutical formulation orkit for use in lowering blood glucose level.

In one aspect, the invention provides a method of treating diabetesmellitus in a subject in need thereof comprising administering apharmaceutical formulation of the invention.

In one aspect, the invention provides a method of treating hyperglycemiain a subject in need thereof comprising administering a pharmaceuticalformulation of the invention.

In one aspect, the invention provides a method of lowering blood glucoselevels in a subject in need thereof comprising administering apharmaceutical formulation of the invention.

In one aspect, the invention provides a medical device for administeringa pharmaceutical formulation of the invention to an animal and/or human.

The present invention is illustrated by the following Examples. However,it should be understood that the present invention is not limited to thespecific details of these examples.

EXAMPLES Example 1

Manufacturing Process

(a) Polysorbate Solution

Polysorbate Solution was prepared by dissolving 1.00 g polysorbate 20 inwater for injection (according to Ph. Eur.) and by filling up with waterfor injection to final volume of 1000 mL.

(b) Zinc Chloride Solution

Zinc Chloride Solution (containing Zn(II)) was prepared by dissolving2.00 g zinc chloride in water for injection and by filling up with waterfor injection to final volume of 1000 mL.

(c) Solution A

The final composition of Solution A is given in Table 1:

TABLE 1 Composition of Solution A Com- position Composition perComposition per Excipient per 200 mL 400 mL 1000 mL 1. Sodium chloride 6.8 g 13.60 g 34.00 g 2. Phenol  1.5 g   3.0 g   7.5 g 3. m-Cresol 1.72g  3.44 g   8.6 g 4. Sodium hydroxide ad pH 8.65 ad pH 8.65 ad pH 8.65solution (rounded: (rounded: (rounded: pH 9.0) pH 9.0) pH 9.0) 5.Hydrochloric acid ad pH 8.65 ad pH 8.65 ad pH 8.65 (rounded: (rounded:(rounded: pH 9.0) pH 9.0) pH 9.0) 6. Water for Injection ad 200 mL = ad400 mL = ad 1000 mL = 205.2 g 410.4 g 1026 g

Solution A was prepared as described in the following:

-   -   1. It was started with approximately 500 mL water for injection.    -   2. 34.00 g sodium chloride, 7.5 g phenol and 8.6 g m-cresol were        dissolved while stirring constantly.    -   3. Solution was filled up to approximately 900 g with water for        injection.    -   4. Solution was stirred for approximately 15 min using a        magnetic stirrer.    -   5. pH was checked (pH should be 8.65, rounded: pH 9.0). If pH        value is not 8.65, the pH was adjusted to said range using        hydrochloric acid 1 N or sodium hydroxide solution 1 N.    -   6. Solution was filled up to 1026 g with water for injection.

(d) Final Solution

The final composition of Final Solution is given in Table 2:

TABLE 2 Composition of Final Solution Com- position Composition perComposition per Excipient per mL 1000 mL 2000 mL 1. Insulin aspart 3.496mg   3.496 g   6.992 g (rounded: (rounded: (rounded: 3.5 mg) 3.5 g) 7.0g) 2. ZnCl₂ 40.87 μg 0.04087 g 0.08174 g 3. Sodium chloride  6.80 mg   6.80 g   13.60 g 4. Phenol  1.50 mg    1.50 g    3.00 g 5. m-Cresol 1.72 mg    1.72 g    3.44 g 6. Polysorbate 20  0.02 mg    0.02 g   0.04 g 7. Sodium hydroxide solution ad pH 7.4 ad pH 7.4 ad pH 7.4 8.Hydrochloric acid ad pH 7.4 ad pH 7.4 ad pH 7.4 9. Water for Injectionad 1 mL = ad 1000 mL = ad 2000 mL = 1.005 g 1005 g 2010 g

In the following, preparation of the composition per 2000 mL isdescribed. Other volumes (e.g. composition per 1000 mL) can be preparedin the same way (using the corresponding amount of insulin aspart andexcipients).

Final Solution was prepared as described in the following:

-   -   1. It was started with approximately 300 mL water for injection        (according to Ph. Eur.).    -   2. 6.992 g (rounded 7.0 g) insulin aspart were added to the 300        mL water for injection while stirring constantly (a suspension        of insulin aspart in water for injection is formed).    -   3. pH value was checked.    -   4. pH value was changed to approximately 3.1 to 3.2 by adding        hydrochloric acid 0.1 N or sodium hydroxide solution 0.02 N to        dissolve the insulin aspart.    -   5. Solution was stirred for approximately 15 min using a        magnetic stirrer.    -   6. 40.866 g Zinc Chloride Solution was added to the solution        while stirring constantly.    -   7. 40 g Polysorbate Solution was added while stirring        constantly.    -   8. Solution was filled up to 600 g with water for injection.    -   9. 410.4 g Solution A was added slowly and carefully while        stirring constantly.    -   10. pH was adjusted to 7.4 (range 7.2 to 7.6) using hydrochloric        acid 0.1 N or sodium hydroxide solution 0.02 N.    -   11. Solution was filled up to 2010 g (corresponds to 100% of the        Final Solution).

Quality control: Final solution was a clear and uncolored solution,showed a pH value of 7.4 (plus/minus 0.2; at 20-25° C.).

The Final Solution was applied to sterile filtration using ‘SartoporeMinisart high flow’ filter (filter material: polyethersulfone; poresize: 0.2 μm; supplier: Sartorius).

The Final Solution after sterile filtration was a clear and uncoloredsolution and showed an osmolality of 260 mOsmol/kg (plus/minus 30).

The Final Solution after sterile filtration was filled into appropriatevials (volume: 5 and 10 mL; 13 mm; clear glas; glas type 1).

The vials—containing the Final Solution after sterile filtration—wasstored between +2° C. and +8° C. and protected from light.

Example 2

Control of the Formulation

(a) Analytical Procedures

Tests were carried out using compendial analytical test methods, whereapplicable. The quality control concept has been established taking intoaccount the cGMP requirements as well as the current status of the ICHprocess.

The non-compendial and chromatographic analytical procedures used tocontrol the formulation are summarized in the following:

Description

Visually examined a number of containers for conformance to theacceptance criteria.

Identification (HPLC)

The identity of the active ingredient was ensured by comparing theretention time of the drug formulation sample with the retention time ofthe reference standard using a reverse phase HPLC method. The method wasalso used for the determination of assay of the active ingredient, forthe determination of the related compounds and impurities, and forquantifying the preservatives m-cresol and phenol.

Assay (HPLC)

The test was carried out by reverse phase liquid chromatography (HPLC).The method was also used for the identification, the determination ofassay of the active ingredient, for the determination of the relatedcompounds and impurities, and for quantifying the preservatives m-cresoland phenol. Column: Lichrosorb RP18, particle size 5 μm, pore size 100 Å(250 mm×4.0 mm), thermostated at +35° C. Autosampler: Thermostated at≤+8° C. Mobile phase A: Sodium sulfate solved in water, 14 g/mL,adjusted with phosphoric acid and sodium hydroxide to a pH of 3.4.Mobile phase B: Water/acetonitrile (50:50 v/v). Gradient is shown inTable 3.

TABLE 3 HPLC gradient Time [min] Mobile phase A [%] Mobile phase B [%] 0to 42 57.7 42.3 42 to 47 linear to 20 80 47 to 52 20 80 52 to 53 linearto 57.7 42.3 53 to 60 equilibration 57.7 42.5

Flow rate: 1.0 mL/min. Injection volume: 10 μL. Detection: 214 nm (forthe active ingredient) and 260 nm (for m-cresol and phenol). Typical runtime: 60 min.

Assay of the active ingredient, m-cresol and phenol were calculated byexternal standardization. Impurities were calculated using the peak areapercent method.

Test solution: The formulation was used without any dilution or furthertreatment.

Related Compounds and Impurities (HPLC)

The same chromatographic conditions as for “Assay (H PLC)” were used forthe determination of related compounds and impurities. Related compoundsand Impurities were calculated using the peak area percent method.

High Molecular Weight Proteins (HMWPs)

The high molecular weight proteins were determined using high pressuresize exclusion chromatography (HPSEC). Column: Waters Insulin HMWP,particle size 5-10 μm, pore size 12-12.5 nm (300 mm×7.8 mm),thermostated at room temperature. Autosampler: thermostated at ≤+8° C.Mobile phase: 650 mL of arginine solution (1 g/L) was mixed with 200 mLof acetonitrile and 150 mL of glacial acetic acid. Isocratic elutionFlow rate: 1.0 mL/min. Injection volume: 100 μL.

Detection: 276 nm. Typical run time: 35 min.

HMWPs were calculated using the peak area percent method. Test solution:The formulation was used without any dilution or further treatment.

Antimicrobial Preservative Assay

The same chromatographic conditions as for “Assay (H PLC)” were used forthe determination of assay of m-Cresol and of phenol m-cresol and phenolwere calculated by external standardization.

(b) Validation of Analytical Procedures

The HPLC analytical procedure for the formulation for the determinationof identification, assay, and related compounds and impurities wasvalidated to demonstrate specificity, linearity, limit of detection andlimit of quantification, accuracy, precision and range.

(c) Justification of the Acceptance Criteria

Tests and acceptance criteria, as previously presented, were selectedbased on ICH Q6B and on published monographs, analytical resultsobtained, precision of procedures used, Pharmacopoeial and/or regulatoryguidelines, and were in agreement with the standard limits at this stageof development.

Example 3

Stability of the Formulation

(a) Stability of the Formulation

Stability studies for the formulation were initiated according to thestability protocol summary described in the following table. Thecomposition and manufacturing method of the stability batches wererepresentative of the material. The stability profile was assessed forstorage under long term, accelerated, and stress testing conditionsaccording to ICH guidelines. Samples were packed and stored in glassvials with flanged cap with inserted disc and flip-off lid. Thestability data obtained using this packaging material wererepresentative for the preliminary shelf life and storage direction forboth packaging configurations (10 mL glass vials and 3 mL cartridges).Up to now, 12 months stability data are available from a batch filledinto 10 mL vials and 12 months of a batch filled into 3 mL cartridgesongoing stability studies of the formulation.

TABLE 4 Storage Conditions Storage Condition Sampling IntervalsContainer Long Term +5° C. ± 3° C. 1, 2, 3, 6, 9, and 12 months 10 mLvials +5° C. ± 3° C. 1, 2, 3, 6, 9, 12, 18, 24 and 3 mL vials 36 monthsAccelerated +25° C. ± 2° C./ 1, 2, 3, and 6 months 10 mL vials and 60% ±5% RH 3 mL cartridges Stress +40° C. ± 5° C./ 1 month 10 mL vials and75% ± 5% RH 3 mL cartridges Photostability Sun test according to 1 day 3mL cartridges ICH guidelines* Indoor light** 14 days 3 mL cartridges*Overall illumination of not less than 1.2 million lux hours and anintegrated near ultraviolet energy of not less than 200 watt hours/m2. Adark control sample is stored under the same conditions to eliminate anyeffects due to local temperature changes **Variolux, Heraeus, standardfluorescent tubes, GE-Lightening, Type F40/33, irradiance approximately8 W/m2, 2000 Lux. A dark control sample is stored under the sameconditions to evaluate any effects due to local temperature changes

The following tests were performed during stability testing: appearance,assay, related impurities, high molecular weight proteins, pH,particulate matter (visible and subvisible particles), assay ofantimicrobial preservatives (m-cresol and phenol), content of zinc. Theinvestigations on physical and chemical properties after 12 months ofstorage at the long term storage condition of +5° C. confirm thestability of the formulation when stored at the recommended storagecondition. Only very slight changes of the related impurities could beobserved.

When stored at accelerated conditions for 3 months at +25° C./60% RH therelated impurities and high molecular weight proteins increased, howeverstayed within the current acceptance limit. When stored at stressconditions (1 month at +40° C./75% RH) one of the related impuritiesincreased above the acceptance criterion. The content of the activeingredient, m-cresol and phenol remained basically unchanged underaccelerated conditions.

Due to the present results of the stability studies of the formulation,the chemical and physical stability of the formulation can be confirmed.

Tables 5-12 show the long term stability results, wherein batch no.“_0105” is referring to a formulation according to the present inventionfilled into 10 mL vials and wherein batch no. “_318” refers to aformulation according to the present invention filled into 3 mLcartridges.

(b) Comparison of Stability of the Buffer-Free Formulation AgainstBuffer Formulations

A buffered formulation containing a phosphate buffer showed physicalinstability by formation of anorganic particles (formation of sodiumzinc phosphate hydrate, Na₆(ZnPO₄)₆.8 H₂O, Sodalite-type crystalstructure). Another buffer formulation containing citrate buffer showedphysical instability as well, by turning turbid after exposing to slightphysical stress. A buffered formulation containing2-amino-2-hydroxymethyl-propane-1,3-diol (synonym: TRIS) showed anincrease of the related impurities when stored under acceleratedconditions. Additionally, the buffered formulation containing2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) showed three additionalimpurities (identified by LC/MS) when stored under stress conditions(+40° C./75% RH), i.e. a TRIS-adduct+103 Da; a correspondingDesamido-adduct+104 Da; and a Formaldehyde-adduct+12 Da (see FIG. 1 ).The formulation according to the invention does not show theseimpurities.

The relevant items tested and the analytical results after one monthstability, at long term, accelerated and stress conditions regarding thephysico-chemical stability in comparison to the formulation according tothe invention are listed in Table 13.

After storage of the formulations at long-term storage conditions (1month at +5° C.), the formulations were applied to mechanical stress(shaking) and thermal stress (+37° C.). The turbidity was measured(nephelometric investigation).

The stability of the buffer-free formulation (i.e., the alternativeaqueous pharmaceutical formulation comprising at least one insulinanalogue and/or insulin derivative comprising sodium chloride andwithout any additional buffering agent) shows an excellent chemical andphysical stability which qualifies said aqueous pharmaceuticalformulation as medicinal product having a defined shelf life.

TABLE 5 Long term stability +5° C.—batch_0105 Acceptance Time [months]Test item criteria Initial 1 2 3 6 9 12 Appearance of solution (visual)Clarity Monitoring <I (water <I (water <I (water <I (water <I (water <I(water <I (water clear) clear) clear) clear) clear) clear) clear) ColorMonitoring B9 B9 B9 B9 B9 B9 <B9 Assay in insulin 90.0 to 110.0 104.7104.5 insulin 104.3 insulin 104.9 insulin 104.9 insulin 103.1 103.0aspart units insulin insulin insulin (HPLC) insulin aspart aspart aspartaspart aspart aspart aspart aspart units/mL units/mL units/mL units/mLunits/mL units/mL units/mL units/mL Related Compounds(HPLC) B28isoAsp<2.5% 0.20% 0.29% 0.36% 0.42% 0.53% 0.63% 0.79% insulin aspart A21 AspMonitoring 1.29% 1.14% 1.09% 1.14% 1.19% 1.23% 0.44% insulin aspart B3Asp Monitoring <RT <RT <RT <RT <RT <RT 0.24% insulin aspart B3 iso AspMonitoring 0.08% 0.09% 0.10% 0.13% 0.14% 0.30% 0.14% insulin aspartTotal of A21Asp ≤5.0% 1.37% 1.23% 1.19% 1.27% 1.32% 2.16% 1.61% insulinaspart, B3Asp insulin aspart and B3isoAsp insulin aspart Any other ≤2.0%0.69% 0.73% 0.59% 0.65% 0.83% 0.58% 0.46% impurity Total of other ≤3.5%0.87% 0.92% 0.75% 0.84% 0.91% 0.81% 0.62% impurities High molecular≤1.5% 0.24% 0.24% 0.26% 0.28% 0.31% 0.33% 0.32% weight proteins (HPSEC)pH Between 7.0 to 7.8 7.33 7.33 7.35 7.34 7.31 7.38 7.49 Particulatematter Practically free complies complies complies complies compliescomplies Complies (visible particles) from visible particles Assaym-cresol 1.55 to 1.89 1.72 mg/mL 1.72 mg/mL 1.71 mg/mL 1.69 mg/mL 1.69mg/mL 1.68 mg/mL 1.71 mg/mL mg/mL (100.0%) (100.0%) (99.2%) (98.3%)(98.3%) (97.7%) (99.2%) (90.0% to 110.0% of label claim) Assay phenol1.35 to 1.65 1.49 mg/mL 1.48 mg/mL 1.49 mg/mL 1.47 mg/mL 1.47 mg/mL 1.48mg/mL 1.49 mg/mL mg/mL (99.5%) (98.6%) (99.3%) (98.0%) (98.0%) (98.6%)(99.5%) (90.0% to 110.0% of label claim) Zinc (AAS) <40 μg per 100 19.7μg Not tested Not tested Not tested Not tested Not tested 19.5 pg unitsinsulin aspart per 100 per 100 units insulin units insulin aspart aspart(20.6 pg/mL) (20.1 μg/mL) Nephelometry Monitoring 0.77 Not tested 0.77Not tested <I (water Turbid clear) after 3 days

TABLE 6 Accelerated stability +25° C./60%RH—batch_0105 Acceptancecriteria for Time Test item clinical trials Initial results 1 month 2months 3 months 6 months Appearance of solution (visual) Clarity anddegree Monitoring <I (water clear) <l (water clear) <I (water clear) <I(water clear) <I (water clear) of opalescence Degree of colorationMonitoring B9 B9 B9 B9 B9 Assay insulin aspart 90.0 insulin aspart 104.6units/mL 104.6 units/mL 104.3 units/mL 103.7 units/mL 101.8 units/mLunits (HPLC) units/mL to 110.0 insulin aspart units/mL Relatedimpurities (HPLC) B28isoAsp insulin aspart ≤2.5% 0.20% 0.84% 1.49% 2.24%4.11% Total of A21Asp insulin ≤5.0% 1.37% 1.50% 1.74% 2.14% 2.90%aspart, B3Asp insulin aspart and B3isoAsp insulin aspart Any otherunspecified, ≤2.0% 0.69% 0.88% 0.93% 1.26% 2.16% unidentified impurityTotal of other impurities ≤3.5% 0.87% 1.08% 1.11% 1.50% 2.40% Highmolecular weight ≤1.5% 0.24% 0.35% 0.48% 0.67% 1.07% proteins (HPSEC) pH7.0 to 7.8 7.3 7.3 7.4 7.3 7.2 Particulate matter Practically free ofconforms conforms conforms conforms conforms (visible particles) visibleparticles Particulate matter Number of particles 33 not tested nottested not tested 3 (subvisible particles) per container ≥10 μm: ≤6000 10 ≥25 μm: ≤600 Assay m-cresol 1.55 to 1.89 [mg/mL] 1.72 mg/mL 1.72 mg/mL1.71 mg/mL 1.70 mg/mL 1.69 mg/mL Assay phenol 1.35 to 1.65 [mg/mL] 1.49mg/mL 1.48 mg/mL 1.49 mg/mL 1.47 mg/mL 1.47 mg/mL Zinc (AAS) <40 μg per100 units 19.7 μg/100U not tested not tested not tested testing ongoinginsulin aspart

TABLE 7 Stress stability +40° C./75% RH-batch _0105 Acceptance criteriafor Time Test item clinical trials Initial result 1 months Appearance ofsolution (visual) Clarity and degree of opalescence Monitoring <I (waterclear) <I (water clear) Degree of coloration Monitoring B9 B9 Assayinsulin aspart units (HPLC) 90.0 insulin aspart units/mL to 104.6units/mL 97.1 units/mL 110.0 insulin aspart units/mL Related impurities(HPLC) B28isoAsp insulin aspart ≤2.5% 0.20% 4.44% Total of A21Aspinsulin aspart, B3Asp ≤5.0% 1.37% 2.81% insulin aspart and B3isoAspinsulin aspart Any other unspecified, unidentified ≤2.0% 0.69% 2.62%impurity Total of other impurities ≤3.5% 0.87% 2.97% High molecularweight proteins (HPSEC) ≤1.5% 0.24% 1.28% pH 7.0 to 7.8 7.3 7.3Particulate matter (visible particles) Practically free of visibleparticles conforms conforms Particulate matter (subvisible particles)Number of particles per container ≥10 μm: ≤6000 33 8 ≥25 μm: ≤600 1 1Assay m-cresol 1.55 to 1.89 [mg/mL] 1.72 mg/mL 1.70 mg/mL Assay phenol1.35 to 1.65 [mg/mL] 1.49 mg/mL 1.47 mg/mL Zinc (Zn(II)) (AAS) <40 μgper 100 units insulin aspart 19.7 μg/100 U 20.9 μg/100 U

TABLE 8 Long term stability +5° C.—batch_318—up to 12 monthsContainer/closure: 3 mL cartridges Storage condition: +5° C. ±3° C.Storage orientation: Horizontal Time Test item Acceptance criteriaInitial results 1 month 3 months 6 months 9 months 12 months Appearanceof solution (visual) Clarity Monitoring <I (water <I (water <I (water <I(water <I (water <I (water clear) clear) clear) clear) clear) clear)Color Monitoring B9 B9 B9 B9 B9 B9 Assay in insulin aspart 90.0 to 110.0100.9 insulin 100.5 insulin 99.9 insulin 102.6 insulin 97.9 insulin100.2 insulin units (HPLC) ^(a) insulin aspart aspart aspart aspartaspart aspart aspart units/mL units/mL units/mL units/mL units/mLunits/mL units/mL Related compounds(HPLC) B28isoAsp insulin aspart ≤2.5%0.22% 0.40% 0.45% 0.59% 0.61% 0.74% Total of A21Asp insulin aspart,≤5.0% 0.68% 1.01% 1.04% 1.11% 0.62% 0.80% B3Asp insulin aspart andB3isoAsp insulin aspart Any other unspecified, unidentified ≤2.0% 0.45%0.62% 0.65% 0.67% 0.87% 0.34% impurity Total of other impurities ≤3.5%0.53% 0.72% 0.74% 0.84% 0.99% 0.53% High molecular weight ≤1.5% 0.19%0.24% 0.24% 0.23% 0.22% 0.26% proteins (HPSEC) PH 7.0 to 7.8 7.31 7.377.41 7.20 7.39 7.35 Sterility Complies complies complies Particulatematter Practically free complies complies complies complies compliescomplies (visible particles) from visible particles Particulate matter(subvisible particles) Number of particles <6000 60 not tested nottested not tested not tested 213 per container ≥10 μm: Number ofparticles <600 0 0 per container ≥25 μm: Antimicrobial preservativeassay m-cresol 1.55 mg/mL to 1.69 mg/mL 1.70 mg/mL 1.69 mg/mL 1.70 mg/mL1.67 mg/mL 1.69 mg/mL 1.89 mg/mL (98.3%) (98.8%) (98.3%) (101.2%)(97.2%) (98.4%) (90.0% to 110.0% of label claim) phenol 1.35 mg/mL to1.48 mg/mL 1.49 mg/mL 1.49 mg/mL 1.48 mg/mL 1.46 mg/mL 1.49 mg/mL 1.65mg/mL (98.7%) (99.3%) (99.3%) (98.7%) (97.2%) (99.4%) (90.0% to 110.0%of label claim) Zinc (AAS) <40 μg/100 units 19.4 μg/100 not tested nottested not tested not tested not tested insulin aspart units insulinaspart (19.6 μg/mL) Preservative efficacy Complies with Complies withComplies with Ph. Eur./USP Ph.Eur criteria Ph.Eur criteria A and with Aand with USP USP ^(a) The content of insulin aspart is calculatedtogether with B28isoAsp insulin aspart, A21Asp insulin aspart, B3Aspinsulin aspart, and B3isoAsp insulin aspart. 1 unit is equivalent to0.0350 mg of insulin aspart

TABLE 9 Long term stability +5° C.-batch _318-18 to 36 monthsContainer/closure: 3 mL cartridges Storage condition: +5° C. ± 3° C.Storage orientation: Horizontal Time Test item Acceptance criteriaInitial results 18 months 24 months 36 months Appearance of solution(visual) Clarity Monitoring <I (water clear) not yet available not yetavailable not yet available Color Monitoring B9 not yet available notyet available not yet available Assay in insulin aspart units (HPLC)^(a) 90.0 to 110.0 insulin aspart 100.9 insulin aspart not yet availablenot yet available not yet available units/mL units/mL Relatedcompounds(HPLC) B28isoAsp insulin aspart ≤2.5% 0.22% not yet availablenot yet available not yet available Total of A21Asp insulin aspart,≤5.0% 0.68% not yet available not yet available not yet available B3Aspinsulin aspart and B3isoAsp insulin aspart Any other unspecified,unidentified ≤2.0% 0.45% not yet available not yet available not yetavailable impurity Total of other impurities ≤3.5% 0.53% not yetavailable not yet available not yet available High molecular weightproteins (HPSEC) ≤1.5% 0.19% not yet available not yet available not yetavailable PH 7.0 to 7.8 7.31 not yet available not yet available not yetavailable Sterility Complies not yet available not yet availableParticulate matter (visible particles) Practically free from visiblecomplies not yet available not yet available not yet available particlesParticulate matter (subvisible particles) Number of particles percontainer ≥10 ≤6000 60 not yet available not yet available not yetavailable μm: Number of particles per container ≥25 ≤600 0 μm:Antimicrobial preservative assay m-cresol 1.55 mg/mL to 1.89 mg/mL 1.69mg/mL not yet available not yet available not yet available (90.0% to110.0% of label (98.3%) claim) phenol 1.35 mg/mL to 1.65 mg/mL 1.48mg/mL not yet available not yet available not yet available (90.0% to110.0% of label (98.7%) claim) Zinc (AAS) <40 μg/100 units insulin 19.4μg/100 units not yet available aspart insulin aspart (19.6 μg/mL)Preservative efficacy Complies with Ph. Eur./USP not yet available notyet available ^(a): The content of insulin aspart is calculated togetherwith B28isoAsp insulin aspart, A21Asp insulin aspart, B3Asp insulinaspart, and B3isoAsp insulin aspart. 1 unit is equivalent to 0.0350 mgof insulin aspart

TABLE 10 Accelerated stability +25° C./60% RH-batch _318Container/closure: 3 mL cartridges Storage condition: +25° C. ± 2°C./60% ± 5% RH Storage orientation: Horizontal Time Test item Acceptancecriteria Initial results 1 month 3 months 6 months Appearance ofsolution (visual) Clarity Monitoring <I (water clear) <I (water clear)<I (water clear) <I (water clear) Color Monitoring B9 B9 B9 B9 Assay ininsulin aspart units (HPLC) ^(a) 90.0 to 110.0 insulin aspart 100.9insulin 100.5 insulin 99.2 insulin 99.8 insulin units/mL aspart units/mLaspart units/mL aspart units/mL aspart units/mL Related compounds(HPLC)B28isoAsp insulin aspart ≤2.5% 0.22% 1.05% 2.34% 4.06% ^(b) Total ofA21Asp insulin aspart, ≤5.0% 0.68% 1.35% 1.97% 2.91% B3Asp insulinaspart and B3isoAsp insulin aspart Any other unspecified, unidentified≤2.0% 0.45% 0.82% 1.15% 1.64% impurity Total of other impurities ≤3.5%0.53% 0.95% 1.36% 2.12% High molecular weight proteins (HPSEC) ≤1.5%0.19% 0.34% 0.51% 0.62% pH 7.0 to 7.8 7.31 7.35 7.39 7.22 Particulatematter (visible particles) Practically free from complies compliescomplies Complies visible particles Particulate matter (subvisibleparticles) Number of particles per container ≥10 ≤6000 60 not tested nottested 164 μm: Number of particles per container ≥25 ≤600 0 0 μm:Antimicrobial preservative assay m-cresol 1.55 mg/mL to 1.89 mg/mL 1.69mg/mL 1.69 mg/mL 1.68 mg/mL 1.67 mg/mL (90.0% to 110.0% of label (98.3%)(98.3%) (97.6%) (97.1%) claim) phenol 1.35 mg/mL to 1.65 mg/mL 1.48mg/mL 1.49 mg/mL 1.49 mg/mL 1.47 mg/mL (90.0% to 110.0% of label (98.7%)(99.3%) (99.3%) (98.0%) claim) Zinc (AAS) <40 μg/100 units 19.4 μg/100units not tested not tested 20.5 μg/100 units insulin aspart insulinaspart insulin aspart (19.6 μg/mL) (20.5 μg/mL) ^(a) The content ofinsulin aspart is calculated together with B28isoAsp insulin aspart,A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulinaspart. 1 unit is equivalent to 0.0350 mg of insulin aspart ^(b): OOSresult

TABLE 11 Stress stability +40° C./75% RH-batch _318 Container/closure: 3mL cartridges Storage condition: +40° C. ± 2° C./75% ± 5% RH Storageorientation: Horizontal Time Test item Acceptance criteria Initialresult 1 month Appearance of solution (visual) Clarity Monitoring <I(water clear) <I (water clear) Color Monitoring B9 B9 Assay in insulinaspart units (HPLC) ^(a) 90.0 to 110.0 insulin aspart 100.9 insulinaspart units/mL 98.2 insulin aspart units/mL units/mL Relatedcompounds(HPLC) B28isoAsp insulin aspart ≤2.5% 0.22% 5.05% ^(b) Total ofA21Asp insulin aspart, ≤5.0% 0.68% 2.64% B3Asp insulin aspart andB3isoAsp insulin aspart Any other unspecified, unidentified ≤2.0% 0.45%2.38% ^(b) impurity Total of other impurities ≤3.5% 0.53% 2.79% Highmolecular weight proteins (HPSEC) ≤1.5% 0.19% 1.16% pH 7.0 to 7.8 7.317.24 Particulate matter (visible particles) Practically free fromvisible complies complies particles Particulate matter (subvisibleparticles) Number of particles per container ≥10 ≤6000 60 132 μm: Numberof particles per container ≥25 ≤600 0 0 μm: Antimicrobial preservativeassay m-cresol 1.55 mg/mL to 1.89 mg/mL 1.69 mg/mL 1.68 mg/mL (90.0% to110.0% of label (98.3%) (97.7%) claim) phenol 1.35 mg/mL to 1.65 mg/mL1.48 mg/mL 1.48 mg/mL (90.0% to 110.0% of label (98.7%) (98.7%) claim)Zinc (AAS) <40 μg/100 units 19.4 μg/100 units insulin 20.6 μg/100 unitsinsulin insulin aspart aspart (19.6 μg/mL) aspart (20.2 μg/mL) ^(a): Thecontent of insulin aspart is calculated together with B28isoAsp insulinaspart, A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAspinsulin aspart. 1 unit is equivalent to 0.0350 mg of insulin aspart^(b): OOS result

TABLE 12 Photostability (Sun-test)-batch _318 Container/closure: 3 mLcartridges Storage condition: Photostability according to ICH Q1Bguideline (Sun-test) ICH option 1 Storage orientation: HorizontalResults Exposure 1 day 1.2 million lux hours Test item Acceptancecriteria Initial Dark control 200 W-h/m² Appearance of solution (visual)Clarity Monitoring <I (water clear) <I (water clear) <I (water clear)Color Monitoring B9 B9 B8 Assay in insulin aspart units (HPLC)^(a) 90.0to 110.0 insulin 100.9 insulin 100.3 insulin 94.2 insulin aspartunits/mL aspart units/mL aspart units/mL aspart units/mL Relatedcompounds(HPLC) B28isoAsp insulin aspart ≤2.5% 0.22% 0.35% 0.30% Totalof A21Asp insulin aspart, ≤5.0% 0.68% 0.97% 0.91% B3Asp insulin aspartand B3isoAsp insulin aspart Any other unspecified, unidentified ≤2.0%0.45% 0.70% 5.05% ^(b) impurity Total of other impurities ≤3.5% 0.53%0.77% 5.54% ^(b) High molecular weight proteins (HPSEC) ≤1.5% 0.19%0.22% 3.48% ^(b) pH 7.0 to 7.8 7.31 7.30 7.24 Particulate matter(visible particles) Practically free from visible complies compliescomplies particles Particulate matter (subvisible particles) Number ofparticles per container ≥10 ≤6000 60 123 132 μm: Number of particles percontainer ≥25 ≤600 0 0 0 μm: Antimicrobial preservative assay m-cresol1.55 mg/mL to 1.89 mg/mL 1.69 mg/mL 1.70 mg/mL 1.69 mg/mL (90.0% to110.0% of label claim) (98.3%) (98.8%) (98.3%) phenol 1.35 mg/mL to 1.65mg/mL 1.48 mg/mL 1.48 mg/mL 1.48 mg/mL (90.0% to 110.0% of label claim)(98.7%) (98.7%) (98.7%) Photostability (indoor light)-batch _318Container/closure: 3 mL cartridges Storage condition: Photostabilityindoorlight Storage orientation: Horizontal Results Exposure 14 days2000 lux hours Test item Acceptance criteria Initial Dark control 8W-h/m² Appearance of solution (visual) Clarity Monitoring <I (waterclear) <I (water clear) <I (water clear) Color Monitoring B9 B9 B9 Assayin insulin aspart units 90.0 to 110.0 insulin 100.9 insulin 100.3insulin 97.6 insulin (HPLC)^(a) aspart units/mL aspart units/mL aspartunits/mL aspart units/mL Related compounds(HPLC) B28isoAsp insulinaspart ≤2.5% 0.22% 0.45% 0.41% Total of A21Asp insulin aspart, ≤5.0%0.68% 1.06% 1.06% B3Asp insulin aspart and B3isoAsp insulin aspart Anyother unspecified, ≤2.0% 0.45% 0.68% 2.51% ^(b) unidentified impurityTotal of other impurities ≤3.5% 0.53% 0.78% 2.88% High molecular weightproteins ≤1.5% 0.19% 0.23% 0.23% (HPSEC) PH 7.0 to 7.8 7.31 7.27 7.28Particulate matter (visible Practically free from complies compliescomplies particles) visible particles Particulate matter (subvisibleparticles) Number of particles per ≤6000 60 69 410 container ≥10 μm:Number of particles per ≤600 0 0 0 container ≥25 μm: Antimicrobialpreservative assay m-cresol 1.55 mg/mL to 1.69 mg/mL 1.69 mg/mL 1.69mg/mL 1.89 mg/mL (98.3%) (98.3%) (98.3%) (90.0% to 110.0% of labelclaim) phenol 1.35 mg/mL to 1.48 mg/mL 1.48 mg/mL 1.48 mg/mL 1.65 mg/mL(98.7%) (98.7%) (98.7%) (90.0% to 110.0% of label claim) ^(a)The contentof insulin aspart is calculated together with B28isoAsp insulin aspart,A21Asp insulin aspart, B3Asp insulin aspart, and B3isoAsp insulinaspart. ^(b): OOS result

TABLE 13 Comparison of stability of the formulation according to theinvention (_0105) against other formulations Related impurities Total ofA21Asp, Total of Composition B3Asp and other Form- Storage MethioninPolysorbate m-Cresol Phenol ZnCl NaCl B28isoAsp B3isoAsp impuritiesHMWPs Visual Nephelometry ulation condition [mg/mL] 20 [mg/mL] [mg/mL][mg/mL] [μg/mL] [mg/mL] [%] [%] [%] [%] inspection (+37° C./120 rpm)_0062 +5° C. — 0.02 2.1 1.5 40.87 6.8 0.36 1.32 0.78 0.28 conforms clearafter 10 days +25° C./60% RH — 0.02 2.1 1.5 40.87 6.8 1.05 1.63 0.940.41 conforms not performed +40° C./75% RH — 0.02 2.1 1.5 40.87 6.8 4.622.91 2.90 1.2 conforms not performed _0105 +5° C. — 0.02 1.72 1.5 40.876.8 0.29 1.23 0.92 0.24 conforms clear after 10 days +25° C./60% RH —0.02 1.72 1.5 40.87 6.8 0.84 1.50 1.08 0.35 conforms not performed +40°C./75% RH — 0.02 1.72 1.5 40.87 6.8 4.44 2.81 2.97 1.3 conforms notperformed _0063 +5° C. — — 2.1 1.5 40.87 6.8 0.34 1.26 0.84 0.26conforms clear after 10 days +25° C./60% RH — — 2.1 1.5 40.87 6.8 1.021.60 0.97 0.38 conforms not performed +40° C./75% RH — — 2.1 1.5 40.876.8 4.61 2.86 2.78 1.3 conforms not performed _0106 +5° C. — — 1.72 1.540.87 6.8 0.30 1.17 0.85 0.21 conforms clear after 10 days +25° C./60%RH — — 1.72 1.5 40.87 6.8 0.85 1.52 1.07 0.32 conforms not performed+40° C./75% RH — — 1.72 1.5 40.87 6.8 4.47 2.79 2.81 1.1 conforms notperformed _0071 +5° C. 3 0.02 2.1 1.5 40.87 6.2 0.33 1.18 0.95 0.24conforms clear after 10 days +25° C./60% RH 3 0.02 2.1 1.5 40.87 6.20.90 1.56 1.35 0.33 does not not performed conform +40° C./75% RH 3 0.022.1 1.5 40.87 6.2 4.62 2.81 3.59 1.1 conforms not performed _0072 +5° C.3 — 2.1 1.5 40.87 6.2 0.33 1.18 1.02 0.24 conforms clear after 10 days+25° C./60% RH 3 — 2.1 1.5 40.87 6.2 0.90 1.53 1.46 0.33 conforms notperformed +40° C./75% RH 3 — 2.1 1.5 40.87 6.2 4.61 3.00 3.68 1.1conforms not performed Related impurities Total of A21Asp, Total ofComposition B3Asp and other Nephelometry Form- Storage MethioninPolysorbate m-Cresol Phenol TRS ZnCl NaCl B28isoAsp B3isoAsp impuritiesHMWPs Visual (+37° C./ 120 rpm) ulation condition [mg/mL] 20 [mg/mL][mg/mL] [mg/mL] [mg/mL] [μg/mL] [mg/mL] [%] [%] [%] [%] inspection _0060+5° C. — 0.02 2.1 1.5 6 40.87 4.5 0.36 1.27 0.77 0.24 conforms clearafter 10 days +25° C./60% RH — 0.02 2.1 1.5 6 40.87 4.5 1.08 1.62 1.020.34 conforms not performed +40° C./75% RH — 0.02 2.1 1.5 6 40.87 4.54.70 3.14 3.68 1.1 conforms not performed _0066 +5° C. — 0.02 1.72 1.5 640.87 4.5 0.34 1.27 0.80 0.22 conforms clear after 10 days +25° C./60%RH — 0.02 1.72 1.5 6 40.87 4.5 1.03 1.57 1.01 0.30 conforms notperformed +40^(o)C/75% RH — 0.02 1.72 1.5 6 40.87 4.5 4.76 3.09 3.46 1.2conforms not performed _0061 +5° C. — — 2.1 1.5 6 40.87 4.5 0.37 1.250.80 0.22 conforms clear after 10 days +25° C./60% RH — — 2.1 1.5 640.87 4.5 1.08 1.47 1.10 0.31 conforms not performed +40° C./75% RH — —2.1 1.5 6 40.87 4.5 4.73 3.10 3.68 1.1 conforms not performed _0104 +5°C. — — 1.72 1.5 6 40.87 4.5 0.33 1.18 0.76 0.20 conforms clear after 10days +25° C./60% RH — — 1.72 1.5 6 40.87 4.5 0.87 1.55 1.08 0.27conforms not performed +40° C./75% RH — — 1.72 1.5 6 40.87 4.5 4.57 3.053.45 1.1 conforms not performed _0069 +5° C. 3 0.02 2.1 1.5 6 40.87 3.30.33 1.25 0.89 0.22 conforms clear after 10 days +25° C./60% RH 3 0.022.1 1.5 6 40.87 3.3 0.90 1.64 1.26 0.28 conforms not performed +40°C./75% RH 3 0.02 2.1 1.5 6 40.87 3.3 4.64 3.27 3.41 1.0 conforms notperformed _0070 +5° C. 3 — 2.1 1.5 6 40.87 3.3 0.33 1.24 0.97 0.21conforms clear after 10 days +25° C./60% RH 3 — 2.1 1.5 6 40.87 3.3 0.891.63 1.28 0.27 conforms not performed +40° C./75% RH 3 — 2.1 1.5 6 40.873.3 4.61 3.29 3.40 1.0 conforms not performed Related impuritiesComposition Total of Total of Glycerol A21Asp, other Nephelometry Form-Storage 85% Methionin Polysorbate m-Cresol Phenol TRS ZnCl NaClB28isoAsp B3Asp and impurities HMWPs Visual (+37° C./ ulation condition[mg/mL] [mg/mL] 20 [mg/mL] [mg/mL] [mg/mL] [mg/mL] [μg/mL] [mg/mL] [%]B3isoAsp [%] [%] [%] inspection 120 rpm) _0067 +5° C. — — 0.02 2.1 1.51.88 40.87 6.2 0.22 1.36 0.84 0.25 does not suspended conform particlesat to +25° C./60% RH — — 0.02 2.1 1.5 1.88 40.87 6.2 0.81 1.72 1.06 0.36does not not performed conform +40° C./75% RH — — 0.02 2.1 1.5 1.8840.87 6.2 4.46 3.22 2.73 1.3 does not not performed conform _0068 +5° C.— — — 2.1 1.5 1.88 40.87 6.2 0.23 1.41 0.84 0.23 conforms suspendedparticles at to +25° C./60% RH — — — 2.1 1.5 1.88 40.87 6.2 0.81 1.680.97 0.35 conforms not performed +40° C./75% RH — — — 2.1 1.5 1.88 40.876.2 4.49 3.18 2.68 1.2 conforms not performed _0075 +5° C. — 3 0.02 2.11.5 1.88 40.87 5 0.24 1.36 0.82 0.23 conforms clear after 10 days +25°C./60% RH — 3 0.02 2.1 1.5 1.88 40.87 5 0.85 1.69 1.05 0.31 conforms notperformed +40° C./75% RH — 3 0.02 2.1 1.5 1.88 40.87 5 4.47 3.17 3.061.0 conforms not performed _0076 +5° C. — 3 — 2.1 1.5 1.88 40.87 5 0.251.33 0.84 0.21 conforms clear after 10 days +25° C./60% RH — 3 — 2.1 1.51.88 40.87 5 0.83 1.66 1.06 0.30 conforms not performed +40° C./75% RH —3 — 2.1 1.5 1.88 40.87 5 4.41 3.20 2.94 1.0 conforms not performed _0151+5° C. 18.82 — — 1.72 1.5 1.88 40.87 0.58 0.30 1.10 0.98 0.38 conformsprecipitated after 7 days * +25° C./60% RH 18.82 — — 1.72 1.5 1.88 40.870.58 0.91 1.70 1.32 0.55 conforms not performed +40° C./75% RH 18.82 — —1.72 1.5 1.88 40.87 0.58 5.07 5.05 4.95 2.0 conforms not performedRelated impurities Total of A21Asp, Total of Composition B3Asp and otherNephelometry Form- Storage Methionin Polysorbate m-Cresol Phenol TRSZnCl NaCl B28isoAsp B3isoAsp impurities HMWPs Visual (+37° C./ ulationcondition [mg/mL] 20 [mg/mL] [mg/mL] [mg/mL] [mg/mL] [μg/mL] [mg/mL] [%][%] [%] [%] inspection 120 rpm) _0064 +5° C. — 0.02 2.1 1.5 4 40.87 5.60.28 1.34 0.84 0.23 conforms suspended particles after 7 days +25°C./60% RH — 0.02 2.1 1.5 4 40.87 5.6 0.98 1.67 0.98 0.33 conforms notperformed +40^(o)C/75% RH — 0.02 2.1 1.5 4 40.87 5.6 4.63 3.48 2.93 1.1conforms not performed _0107 +5° C. — 0.02 1.72 1.5 4 40.87 5.6 0.271.28 0.89 0.24 conforms clear after 10 days +25° C./60% RH — 0.02 1.721.5 4 40.87 5.6 0.83 1.60 1.03 0.31 conforms not performed +40^(o)C/75%RH — 0.02 1.72 1.5 4 40.87 5.6 4.31 3.30 2.55 1.0 conforms not performed_0065 +5° C. — — 2.1 1.5 4 40.87 5.6 0.29 1.36 0.90 0.22 conforms turbidafter 3 days +25° C./60% RH — — 2.1 1.5 4 40.87 5.6 0.99 1.66 0.99 0.31conforms not performed +40° C./75% RH — — 2.1 1.5 4 40.87 5.6 4.66 3.452.92 1.1 conforms not performed _0108 +5° C. — — 1.72 1.5 4 40.87 5.60.25 1.31 0.82 0.22 conforms turbid after 3 days +25° C./60% RH — — 1.721.5 4 40.87 5.6 0.81 1.62 1.00 0.30 conforms not performed +40° C./75%RH — — 1.72 1.5 4 40.87 5.6 4.29 3.26 2.59 0.91 does not not performedconform _0073 +5° C. 3 0.02 2.1 1.5 4 40.87 4.4 0.33 1.24 0.84 0.24conforms clear after 10 days +25° C./60% RH 3 0.02 2.1 1.5 4 40.87 4.40.90 1.64 1.12 0.31 conforms not performed +40° C./75% RH 3 0.02 2.1 1.54 40.87 4.4 4.43 3.72 3.57 1.2 conforms not performed _0074 +5° C. 3 —2.1 1.5 4 40.87 4.4 0.33 1.19 0.84 0.22 does not turbid after 3 daysconform +25° C./60% RH 3 — 2.1 1.5 4 40.87 4.4 0.92 1.62 1.12 0.29conforms not performed +40° C./75% RH 3 — 2.1 1.5 4 40.87 4.4 4.43 3.723.657 1.2 does not not performed *data taken after 3 months storageunder long term conditions (+5° C.)

1. A pharmaceutical formulation comprising (a). at least one analogueand/or derivative of insulin; (b). Zn(II); (c). sodium chloride; and(d). optionally protamine; wherein the pharmaceutical formulation has apH value in the range from 6.0 to 9.0 and is free of any additionalbuffering agent.
 2. The pharmaceutical formulation according to claim 1,wherein the pharmaceutical formulation is an aqueous pharmaceuticalformulation.
 3. The pharmaceutical formulation according to claim 1,wherein the pharmaceutical formulation has a pH value in the range from7.0 to 7.8.
 4. The pharmaceutical formulation according to claim 1,wherein the analogue of insulin is selected from the group consisting ofinsulin aspart, insulin lispro and insulin glulisine.
 5. Thepharmaceutical formulation according to claim 1, wherein the derivativeof insulin is insulin detemir and/or insulin degludec.
 6. Thepharmaceutical formulation according to claim 1, wherein the analogueand/or derivative of insulin is present in a concentration from 10 U/mLto 1000 U/mL.
 7. The pharmaceutical formulation according to claim 1,wherein Zn(II) is present in a concentration from 0.0100 to 0.0600mg/100 U of the analogue and/or derivative of insulin.
 8. Thepharmaceutical formulation according to claim 1, wherein sodium chlorideis present in a concentration from 0.01 to 15 mg/mL.
 9. Thepharmaceutical formulation according to claim 1, wherein sodium chlorideis present in a concentration from 6.8 to 8.3 mg/mL.
 10. Thepharmaceutical formulation according to claim 1, wherein protamine isprotamine sulfate which is present in a concentration from 0.1 to 0.5mg/mL.
 11. The pharmaceutical formulation according to claim 1, whereinthe pharmaceutical formulation is free of any additional buffering agentselected from the group consisting of2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), phosphate, citric acid,citrate, acetic acid, acetate, glycylglycine and methionine.
 12. Thepharmaceutical formulation according to claim 1, wherein thepharmaceutical formulation comprises one or more further activepharmaceutical ingredients.
 13. The pharmaceutical formulation accordingto claim 12, wherein the further active pharmaceutical ingredient is anantidiabetic agent.
 14. The pharmaceutical formulation according toclaim 12, wherein the further active pharmaceutical ingredient is anantidiabetic agent selected from the group consisting of: (a). a GLP-1receptor agonist; (b). a dual GLP-1 receptor/glucagon receptor agonist;(c). human FGF-21; (d). an FGF-21 analogue; (e). an FGF-21 derivative;(f). insulin; (g). human insulin; (h). an analogue of insulin; and (i).a derivative of insulin.
 15. The pharmaceutical formulation according toclaim 1, wherein the pharmaceutical formulation comprises more than oneanalogue and/or derivative of insulin, wherein one analogue and/orderivative of insulin is a fast acting insulin and one analogue and/orderivative of insulin is a long acting insulin.
 16. The pharmaceuticalformulation according to claim 15, wherein the fast acting insulin isone or more insulin selected from the group consisting of insulinaspart, insulin lispro, and insulin glulisine, and wherein the longacting insulin is one or more insulin selected from the group consistingof insulin detemir and insulin degludec.
 17. The pharmaceuticalformulation according to claim 1, wherein the pharmaceutical formulationconsists of: (a). 3.5 mg/mL insulin aspart; (b). 1.72 mg/mL metacresol;(c). 1.50 mg/mL phenol; (d). 0.0196 mg/mL Zn(II); (e). 6.8 mg/mL sodiumchloride; (f). 0.02 mg/mL polysorbate 20; (g). sodium hydroxide and/orhydrochloric acid to adjust the pH to 7.4, and (h). water.
 18. Thepharmaceutical formulation according to claim 1, wherein thepharmaceutical formulation consists of: (a). 3.5 mg/mL insulin aspart;(b). 1.72 mg/mL metacresol; (c). 1.50 mg/mL phenol; (d). 0.0196 mg/mLZn(II); (e). from 6.8 mg/mL to 8.3 mg/mL sodium chloride; (f). 0.02mg/mL polysorbate 20; (g). from 0.1 mg/mL to 0.5 mg/mL protaminesulfate; (h). sodium hydroxide and/or hydrochloric acid to adjust the pHto a pH in the range from 7.1 to 7.6; and (i). water.
 19. A method forpreparing the pharmaceutical formulation according to claim 1, whereinthe components are mixed together in the form of a solution orsuspension, the pH is adjusted to reach a desired pH, and water is addedto reach a final volume.
 20. A kit comprising one or more separatepackages of (a). the pharmaceutical formulation as claimed in claim 1;and (b). a medical device.
 21. A kit comprising one or more separatepackages of (a). the pharmaceutical formulation as claimed in claim 1;and (b). at least one further active pharmaceutical ingredient; (c). andoptionally a medical device.
 22. The kit according to claim 20, whereinthe further active pharmaceutical ingredient is an antidiabetic agent.23. The kit as claimed in claim 20, wherein the further activepharmaceutical ingredient is an antidiabetic agent selected from thegroup consisting of: (a). a GLP-1 receptor agonist; (b). a dual GLP-1receptor/glucagon receptor agonist; (c). human FGF-21; (d). an FGF-21analogue; (e). an FGF-21 derivative; (f). insulin; (g). human insulin;(h). an analogue of insulin; and (i). a derivative of insulin.
 24. Thekit as claimed in claim 20, wherein the kit comprises more than oneanalogue and/or derivative of insulin, wherein one analogue and/orderivative of insulin is a fast acting insulin and one analogue and/orderivative of insulin is a long acting insulin.
 25. The kit according toclaim 24, wherein the fast acting insulin is selected from the groupconsisting of insulin aspart, insulin lispro and insulin glulisine, andwherein the long acting insulin is selected from the group consisting ofinsulin glargin, insulin detemir and insulin degludec.
 26. Thepharmaceutical formulation as claimed in claim 1 for use in thetreatment of diabetes mellitus.
 27. The pharmaceutical formulation asclaimed in claim 1 for use in the treatment of hyperglycemia.
 28. Thepharmaceutical formulation as claimed in claim 1 for use in loweringblood glucose level.
 29. A method of treating diabetes mellitus in asubject in need thereof comprising administering the pharmaceuticalformulation of claim
 1. 30. A method of treating hyperglycemia in asubject in need thereof comprising administering the pharmaceuticalformulation of claim
 1. 31. A method of lowering blood glucose levels ina subject in need thereof comprising administering the pharmaceuticalformulation of claim
 1. 32. A medical device for administering thepharmaceutical formulation as claimed in claim 1 to an animal and/orhuman.